DNA encoding for CSF-1 and accompanying recombinant systems

ABSTRACT

A colony stimulating factor, CSF-1, is a lymphokine useful in overcoming the immunosuppression induced by chemotherapy or resulting from other causes. CSF-1 is obtained in usable amounts by recombinant methods, including cloning and expression of the murine and human DNA sequences encoding this protein.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of application Ser. No. 876,819,filed June 20, 1986 now abandoned which is a continuation-in-part ofU.S. patent application Ser. No. 821,068, filed Jan. 21, 1986, nowabandoned which is a continuation-in-part of U.S. patent applicationSer. No. 756,814, filed July 18, 1985, now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 744,924, filedJune 14, 1985, now abandoned which is a continuation-in-part of U.S.patent application Ser. No. 728,834, filed Apr. 30, 1985, now abandonedwhich is a continuation-in-part of U.S. Ser. No. 698,359, filed Feb. 5,1985, now abandoned.

TECHNICAL FIELD

The present invention relates to the use of recombinant technology forproduction of lymphokines ordinarily produced in low concentration. Morespecifically, the invention relates to the cloning and expression of aDNA sequence encoding human colony stimulating factor-1 (CSF-1).

BACKGROUND ART

The ability of certain factors produced in very low concentration in avariety of tissues to stimulate the growth and development of bonemarrow progenitor cells into granulocytes and/or macrophages has beenknown for nearly 15 years. The presence of such factors in sera, urinesamples, and tissue extracts from a number of species is demonstrableusing an in vitro assay which measures the stimulation of colonyformation by bone marrow cells plated in semi-solid culture medium.There is no known in vivo assay. Because these factors induce theformation of such colonies, the factors collectively have been calledColony Stimulating Factors (CSF).

More recently, it has been shown that there are at least four subclassesof human CSF proteins which can be defined according to the types ofcells found in the resultant colonies. One subclass, CSF-1, results incolonies containing macrophages predominantly. Other subclasses producecolonies which contain both neutrophilic granulocytes and macrophages;which contain predominantly neutrophilic granulocytes; and which containneutrophilic and eosinophilic granulocytes and macrophages.

There are murine factors analogous to the first three of the above humanCSFs. In addition, a murine factor called IL-3 induces colonies frommurine bone marrow cells which contain all these cell types plusmegakaryocytes, erythrocytes, and mast cells, in various combinations.These CSFs have been reviewed by Dexter, T. M., Nature (1984) 309: 746,and Vadas, M. A., et al, J Immunol (1983) 130: 793.

The invention herein is concerned with the recombinant production ofproteins which are members of the first of these subclasses, CSF-1. Thissubclass has been further characterized and delineated by specificradioimmunoassays and radioreceptor assays--e.g., antibodies raisedagainst purified CSF-1 are able to suppress specifically CSF-1 activity,without affecting the biological activities of the other subclasses, andmacrophage cell line J774 contains receptors which bind CSF-1specifically. A description of these assays was published by Das, S. K.,et al, Blood (1981) 58:630.

Purification methods for various CSF proteins have been published andare described in the following paragraphs.

Stanley, E. R., et al, J Biol Chem (1977) 252: 4305 reportedpurification of a CSF protein from murine L929 cells to a specificactivity of about 1×10⁸ units/mg, which also stimulated mainlymacrophage production. Waheed, A., et al, Blood (1982) 60: 238,described the purification of mouse L-cell CSF-1 to apparent homogeneityusing a rabbit antibody column and reported the first 25 amino acids ofthe murine sequence (Ben-Avram, C. M., et al, Proc Natl Acad Sci (USA)(1985) 882: 4486).

Stanley, E. R., et al, J Biol Chem (1977) 252: 4305-4312 disclosed apurification procedure for CSF-1 from human urine and Das, S. K., et al,Blood (1981) 58: 630; J Biol Chem (1982) 257: 13679 obtained a humanurinary CSF-1 at a specific activity of 5×10⁷ units/mg which producedonly macrophage colonies, and outlined the relationship of glycosylationof the CSF-1 proteins prepared from cultured mouse L-cells and fromhuman urine to their activities. Wang, F. F., et al, J Cell Biochem(1983) 21: 263, isolated human urinary CSF-1 to specific activity of 10⁸U/mg. Waheed, A., et al, disclosed purification of human urinary CSF-1to a specific activity of 0.7-2.3×10⁷ U/mg on a rabbit antibody column(Exp Hemat (1984) 12: 434).

Wu, M., et al, J Biol Chem (1979) 254: 6226 reported the preparation ofa CSF protein from cultured human pancreatic carcinoma (MIAPaCa) cellswhich resulted in the growth of murine granulocytic and macrophagiccolonies. The resulting protein had a specific activity of approximately7×10⁷ units/mg.

Partially purified preparations of various CSFs have also been reportedfrom human and mouse lung-cell conditioned media (Fojo, S. S., et al,Biochemistry (1978) 17: 3109; Burgess, A. W., et al, J Biol Chem (1977)252: 1998); from human T-lymphoblast cells (Lusis, A. J., et al, Blood(1981) 57: 13; U.S. Pat. No. 4,438,032); from human placentalconditioned medium to apparent homogeneity and specific activity of7×10⁷ U/mg (Wu, M., et al, Biochemistry (1980) 19: 3846).

A significant difficulty in putting CSF proteins in general, and CSF-1in particular, to any useful function has been their unavailability indistinct and characterizable form in sufficient amounts to make theiremployment in therapeutic use practical or even possible. The presentinvention remedies these deficiencies by providing purified human andmurine CSF-1 in useful amounts through recombinant techniques.

A CSF protein of a different subclass, murine and human GM-CSF has beenpurified and the cDNAs cloned. This protein was shown to be distinctfrom other CSFs, e.g., CSF-1, by Gough, et al, Nature (1984) 309:763-767. Murien IL-3 has been cloned by Fung, M. C., et al, Nature(1984) 307: 233. See also Yokota, T., et al, PNAS (1984) 81: 1070-1074;Wong, G. G., et al, Science (1985) 228: 810-815; Lee, F., et al, PNAS(1985) 82: 4360-4364; and Cantrell, M. A., et al, PNAS (1985) 82:6250-6254.

DISCLOSURE OF THE INVENTION

In one aspect, the present invention relates to recombinant CSF-1protein, including the biologically active proteins containingmodifications of primary amino acid sequence of the native protein.CSF-1 protein in recombinanat form can be obtained in quantity, can bemodified advantageously through regulation of the post-translationalprocessing provided by the host, and can be intentionally modified atthe genetic or protein level to enhance its desirable properties. Forexample, muteins having deletions of substantial portions of the carboxyterminal one-third of the polypeptide are thus active. Thus, theavailability of CSF-1 in recombinant form provides both flexibility andcertain quantitative advantages which make possible applications for useof the protein therapeutically, that are unavailable with respect to thenative protein.

In other aspects, the invention relates to an isolated DNA sequenceencoding recombinant CSF-1, to recombinant expression systems for thissequence and to vectors containing them, to recombinant hosts which aretransformed with these vectors, and to cultures producing therecombinant protein. The invention further relates to methods forproducing the recombinant protein and to the materials significant inits production.

In addition, the invention relates to compositions containing CSF-1which are useful in pharmaceutical and therapeutic applications, and tomethods of use for such compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the partial amino acid sequences of human urinary andmurine L-929 cell CSF-1 as determined from purified native proteins.

FIG. 2 shows the sequence of certain oligomer probes for murine CSF-1.

FIG. 3 shows the sequence of oligomer probes used to obtain humangenomic CSF-1.

FIG. 4 shows the sequenced portion of a 3.9 kb HindIII fragment encodinghuman CSF-1 sequences and the deduced amino acid sequences for the exonregions.

FIG. 5 shows the DNA and deduced amino acid sequences for a cDNA cloneencoding CSF-1.

FIG. 6 shows a comparison of the activities of CSF-1 and other colonystimulating factors in enhancing the ability of macrophages to killtumor cells.

FIG. 7 shows the results of sucrose gradient fractionation of MIAPaCamRNA.

MODES FOR CARRYING OUT THE INVENTION

A. Definitions

"Colony stimulating factor-1 (CSF-1)" refers to a protein which exhibitsthe spectrum of activity understood in the art for CSF-1--i.e., whenapplied to the standard in vitro colony stimulating assay of Metcalf,D., J Cell Physiol (1970) 76: 89, it results in the formation ofprimarily macrophage colonies. Native CSF-1 is a glycosylated dimer;dimerization may be necessary for activity. Contemplated within thescope of the invention and within the definition of CSF-1 are both thedimeric and monomeric forms. The monomeric form may be converted to thedimer by in vitro provision of intracellular conditions, and the monomeris per se useful as an antigen to produce anti-CSF-1 antibodies.

There appears to be some species specificity: Human CSF-1 is operativeboth on human and on murine bone marrow cells; murine CSF-1 does notshow activity with human cells. Therefore, "human" CSF-1 should bepositive in the specific murine radioreceptor assay of Das, S. K., etal, Blood (1981) 58: 630, although there is not necessarily a completecorrelation. The biological activity of the protein will generally alsobe inhibited by neutralizing antiserum to human urinary CSF-1 (Das, S.K., et al, supra). However, in certain special circumstances (such as,for example, where a particular antibody preparation recognizes a CSF-1epitope not essential for biological function, and which epitope is notpresent in the particular CSF-1 mutein being tested) this criterion maynot be met.

Certain other properties of CSF-1 have been recognized more recently,including the ability of this protein to stimulate the secretion ofseries E prostaglandins, interleukin-1, and interferon from maturemacrophages (Moore, R., et al, Science (1984) 223: 178). The mechanismfor these latter activities is not at present understood, and forpurposes of definition herein, the criterion for fulfillment of thedefinition resides in the ability to stimulate the formation ofmonocyte/macrophage colonies using bone marrow cells from theappropriate species as starting materials, under most circumstances (seeabove) the inhibition of this activity by neutralizing antiserum againstpurified human urinary CSF-1, and, where appropriate for species type, apositive response to the radioreceptor assay. (It is known that theproliferative effect of CSF-1 is restricted to cells of mononuclearphagocytic lineage (Stanely, E. R., The Lymphokines (1981), Stewart, W.E., II, et al, ed, Humana Press, Clifton, NJ), pp. 102-132) and thatreceptors for CSF-1 are restricted to these cell lines (Byrne, P. V., etal, Cell Biol (1981) 91: 848)).

As is the case for all proteins, the precise chemical structure dependson a number of factors. As ionizable amino and carboxyl groups arepresent in the molecule, a particular protein may be obtained as anacidic or basic salt, or in neutral form. All such preparations whichretain their activity when placed in suitable environmental conditionsare included in the definition. Further, the primary amino acid sequencemay be augmented by derivatization using sugar moieties (glycosylation)or by other supplementary molecules such as lipids, phosphate, acetylgroups and the like, more commonly by conjugation with saccharides. Theprimary amino acid structure may also aggregate to form complexes, mostfrequently dimers. Indeed, native human urinary CSF-1 is isolated as ahighly glycosylated dimer. Certain aspects of such augmentation areaccomplished through post-translational processing systems of theproducing host; other such modifications may be introduced in vitro. Inany event, such modifications are included in the definition so long asthe activity of the protein, as defined above, is not destroyed. It isexpected, of course, that such modifications may quantitatively orqualitatively affect the activity, either by enhancing or diminishingthe activity of the protein in the various assays.

Further, individual amino acid residues in the chain may be modified byoxidation, reduction, or other derivatization, and the protein may becleaved to obtain fragments which retain activity. Such alterationswhich do not destroy activity do not remove the protein sequence fromthe definition.

Modifications to the primary structure itself by deletion, addition, oralteration of the amino acids incorporated into the sequence duringtranslation can be made without destroying the activity of the protein.Such substitutions or other alterations result in proteins having anamino acid sequence which falls within the definition of proteins"having an amino acid sequence substantially equivalent to that ofCSF-1". Indeed, human and murine derived CSF-1 proteins havenon-identical but similar primary amino acid sequences which display ahigh homology.

For convenience, the mature protein amino acid sequence of the monomericportion of a dimeric protein shown in FIG. 5, deduced from the cDNAclone illustrated herein, is designated mCSF-1 (mature CSF-1). FIG. 5shows the presence of a 32 residue putative signal sequence, which ispresumably cleaved upon secretion from mammalian cells; mCSF-1 isrepresented by amino acids 1-224 shown in that figure. Specificallyincluded in the definition of human CSF-1 are muteins which monomers anddimers are mCSF-1 and related forms of mCSF-1, designated by theirdifferences from mCSF-1. CSF-1 derived from other species may fit thedefinition of "human" CSF-1 by virtue of its display of the requisitepattern of activity as set forth above with regard to human substrate.

Also for convenience, the amino acid sequence of mCSF-1 will be used asa reference and other sequences which are substantially equivalent tothis in terms of CSF-1 activity will be designated by referring to thesequence shown in FIG. 5. The substitution of a particular amino acidwill be noted by reference to the amino acid residue which it replaces.Thus, for example, ser₉₀ CSF-1 refers to the protein which has thesequence shown in FIG. 5 except that the amino acid at position 90 isserine rather than cysteine. Deletions are noted by a ∇ followed by thenumber of amino acids deleted from the N-terminal sequence, or by thenumber of amino acids remaining when residues are deleted from theC-terminal sequence, when the number is followed by a minus sign. Thus,∇₄ CSF-1 refers to CSF-1 of FIG. 5 wherein the first 4 amino acids fromthe N-terminus have been deleted; ∇₁₃₀₋ refers to CSF-1 wherein the last94 amino acids following amino acid 130 have been deleted. Illustratedbelow are, for example, asp₅₉ CSF-1, which contains an aspartic residueencoded by the gene (FIG. 5) at position 59 rather than the tyrosineresidue encoded by the cDNA, and ∇₁₅₈₋ CSF-1, which comprises only aminoacids 1-158 of mCSF-1.

"Operably linked" refers to juxtaposition such that the normal functionof the components can be performed. Thus, a coding sequence "operablylinked" to control sequences refers to a configuration wherein thecoding sequence can be expressed under the control of these sequences.

"Control sequences" refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences which are suitable for procaryotes, for example,include a promoter, optionally an operator sequence, a ribosome bindingsite, and possibly, other as yet poorly understood, sequences.Eucaryotic cells are known to utilize promoters, polyadenylationsignals, and enhancers.

"Expression system" refers to DNA sequences containing a desired codingsequence and control sequences in operable linkage, so that hoststransformed with these sequences are capable of producing the encodedproteins. In order to effect transformation, the expression system maybe included on a vector; however, the relevant DNA may then also beintegrated into the host chromosome.

As used herein "cell", "cell line", and "cell culture" are usedinterchangeably and all such designations include progeny. Thus"transformants" or "transformed cells" includes the primary subject celland cultures derived therefrom without regard for the number oftransfers. It is also understood that all progeny may not be preciselyidentical in DNA content, due to deliberate or inadvertent mutations.Mutant progeny which have the same functionality as screened for in theoriginally transformed cell, are included. Where distinct designationsare intended, it will be clear from the context.

B. General Description

The CSF-1 proteins of the invention are capable both of stimulatingmonocyte-precursor/macrophage cell production from progenitor marrowcells, thus enhancing the effectiveness of the immune system, and ofstimulating such functions of these differentiated cells as thesecretion of lymphokines in the mature macrophages.

In one appplication, these proteins are useful as adjuncts tochemotherapy. It is well understood that chemotherapeutic treatmentresults in suppression of the immune system. Often, although successfulin destroying the tumor cells against which they are directed,chemotherapeutic treatments result in the death of the subject due tothis side effect of the chemotoxic agents on the cells of the immunesystem. Administration of CSF-1 to such patients, because of the abilityof CSF-1 to mediate and enhance the growth and differentiation of bonemarrow-derived precursors into macrophages and monocytes and tostimulate some of the functions of these mature cells, results in arestimulation of the immune system to prevent this side effect, and thusto prevent the propensity of the patient to succumb to secondaryinfection. Other patients who would be helped by such treatment includethose being treated for leukemia through bone marrow transplants; theyare often in an immunosuppressed state to prevent rejection. For thesepatients also, the immunosuppression could be reversed by administrationof CSF-1.

In general, any subject suffering from immunosuppression whether due tochemotherapy, bone marrow transplantation, or other, accidental forms ofimmunosuppression such as disease (e.g., acquired immune deficiecnysyndrome) would benefit from the availability of CSF-1 forpharmacological use. In addition, subjects could be supplied enhancedamounts of previously differentiated macrophages to supplement those ofthe indigenous system, which macrophages are produced by in vitroculture of bone marrow or other suitable preparations treated withCSF-1. These preparations include those of the patient's own bloodmonocytes, which can be so cultured and returned for local or systemictherapy.

The ability of CSF-1 to stimulate production of lymphokines bymacrophages and to enhance their ability to kill target cells also makesCSF-1 directly useful in treatment of neoplasms and infections.

CSF-1 stimulates the production of interferons by murine-derivedmacrophage (Fleit, H. B., et al, J Cell Physiol (1981) 108: 347), andhuman, partially purified, CSF-1 from MIAPaCa cells stimulates thepoly(I):poly (C)-induced production of interferon and TNF from humanmonocytes as illustrated below. In addition, CSF-1 stimulates theproduction of myeloid CSF by human blood monocytes.

Also illustrated below is a demonstration of the ability of murine CSF-1(from L-cell-conditioned medium) to stimulate normal C3H/HeN mouseperitoneal macrophages to kill murine sarcoma TU5 targets. This activityis most effective when the CSF-1 is used as pretreatment and during theeffector phase. The ability of CSF-1 to do so is much greater than thatexhibited by other colony stimulating factors, as shown in FIG. 6hereinbelow. In addition, the ability of murine cells to attack virusesis enhanced by CSF-1.

Murine CSF-1 is inconsistently reported to stimulate murine macrophageto be cytostatic to P815 tumor cells (Wing, E. J., et al, J Clin Invest(1982) 69: 270) or not to kill other leukemia targets (Ralph, P. et al,Cell Immunol (1983) 76: 10). Nogawa, R. T., et al, Cell Immunol (1980)53: 116, report that CSF-1 may stimulate macrophages to ingest and killyeast.

Thus, in addition to overcoming immunosuppression per se, CSF-1 can beused to destroy the invading organisms or malignant cells indirectly bystimulation of macrophage secretions and activity.

The CSF-1 of the invention may be formulated in conventional waysstandard in the art for the administration of protein substances.Administration by injection is preferred; formulations include solutionsor suspensions, emulsions, or solid composition for reconstitution intoinjectables. Suitable excipients include, for example, Ringer'ssolution, Hank's solution, water, saline, glycerol, dextrose solutions,and the like. In addition, the CSF-1 of the invention may bepreincubated with preparations of cells in order to stimulateappropriate responses, and either the entire preparation or thesupernatant therefrom introduced into the subject. As shown hereinbelow,the materials produced in response to CSF-1 stimulation by various typesof blood cells are effective against desired targets, and the propertiesof these blood cells themselves to attack invading viruses or neoplasmsmay be enhanced. The subject's own cells may be withdrawn and used inthis way, or, for example, monocytes or lymphocytes from anothercompatible individual employed in the incubation.

Although the existence of a pattern of activity designated CSF-1 hasbeen known for some time, the protein responsible has never beenobtained in both sufficient purity and in sufficient amounts to permitsequence determination, nor in sufficient purity and quantity to providea useful therapeutic function. Because neither completely pure practicalamounts of the protein nor its encoding DNA have been available, it hasnot been possible to optimize modifications to structure by providingsuch alternatives as those set forth in A above, nor has it beenpossible to utilize this protein in a therapeutic context.

The present invention remedies these defects. Through a variety ofadditional purification procedures, sufficient pure CSF-1 has beenobtained from human urine to provide some amino acid sequence, thuspermitting the construction of DNA oligomeric probes. The probes areuseful in obtaining the coding sequence for the entire protein. Oneapproach, illustrated below, employs probes designed with respect to thehuman N-terminal sequence to probe the human genomic library to obtainthe appropriate coding sequence portion. The human genomic clonedsequence can be expressed directly using its own control sequences, orin constructions appropriate to mammalian systems capable of processingintrons. The genomic sequences are also used as probes for a human cDNAlibrary obtained from a cell line which produces CSF-1 to obtain cDNAencoding this protein. The cDNA, when suitably prepared, can beexpressed directly in COS or CV-1 cells and can be constructed intovectors suitable for expression in a wide range of hosts.

Thus these tools can provide the complete coding sequence for humanCSF-1 from which expression vectors applicable to a variety of hostsystems can be constructed and the coding sequence expressed. Thevariety of hosts available along with expression vectors suitable forsuch hosts permits a choice among post-translational processing systems,and of environmental factors providing conformational regulation of theprotein thus produced.

C. Suitable Hosts, Control Systems and Methods

In general terms, the production of a recombinant form of CSF-1typically involves the following:

First a DNA encoding the mature (used here to include all muteins)protein, the preprotein, or a fusion of the CSF-1 protein to anadditional sequence which does not destroy its activity or to additionalsequence cleavable under controlled conditions (such as treatment withpeptidase) to give an active protein, is obtained. If the sequence isuninterrupted by introns it is suitable for expression in any host. Ifthere are introns, expression is obtainable in mammalian or othereucaryotic systems capable of processing them. This sequence should bein excisable and recoverable form. The excised or recovered codingsequence is then placed in operable linkage with suitable controlsequences in a replicable expression vector. The vector is used totransform a suitable host and the transformed host cultured underfavorable conditions to effect the production of the recombinant CSF-1.Optionally the CSF-1 is isolated from the medium or from the cells;recovery and purification of the protein may not be necessary in someinstances, where some impurities may be tolerated. For example, for invitro cultivation of cells from which a lymphokine factor will beisolated for administration to a subject, complete purity is notrequired. However, direct use in therapy by administration to a subjectwould, of course, require purification of the CSF-1 produced.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences can be obtained by preparingsuitable cDNA from cellular messenger and manipulating the cDNA toobtain the complete sequence. Alternatively, genomic fragments may beobtained and used directly in appropriate hosts. The constructions forexpression vectors operable in a variety of hosts are made usingappropriate replicons and control sequences, as set forth below.Suitable restriction sites can, if not normally available, be added tothe ends of the coding sequence so as to provide an excisable gene toinsert into these vectors.

The control sequences, expression vectors, and transformation methodsare dependent on the type of host cell used to express the gene.Generally, procaryotic, yeast, or mammalian cells are presently usefulas hosts. Since native CSF-1 is secreted as a glycosylated dimer, hostsystems which are capable of proper post-translational processing arepreferred. Accordingly, although procaryotic hosts are in general themost efficient and convenient for the production of recombinantproteins, eucaryotic cells, and, in particular, mammalian cells arepreferred for their processing capacity. Recombinant CSF-1 produced bybacteria would require in vitro dimerization. In addition, there is moreassurance that the native signal sequence will be recognized bymammalian cell hosts making secretion possible, and purificationtherefore easier.

C.1. Control Sequences And Corresponding Hosts

Procaryotes most frequently are represented by various strains of E.coli. However, other microbial strains may also be used, such asbacilli, for example Bacillus subtilis, various species of Pseudomonas,or other bacterial strains. In such procaryotic systems, plasmid vectorswhich contain replication sites and control sequences derived from aspecies compatible with the host are used. For example, E. coli istypically transformed using derivatives of pBR322, a plasmid derivedfrom an E. coli species by Bolivar, et al, Gene (1977) 2: 95. pBR322contains genes for ampicillin and tetracycline resistance, and thusprovides additional markers which can be either retained or destroyed inconstructing the desired vector. Commonly used procaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta-lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al, Nature (1977) 198: 1056) and the tryptophan (trp)promoter system (Goeddel, et al Nucleic Acids Res (1980) 8: 4057) andthe lambda derived P_(L) promoter and N-gene ribosome binding site(Shimatake, et al, Nature (1981) 292: 128), which has been made usefulas a portable control cassette, as set forth in U.S. Ser. No. 685,312now U.S. Pat. No. 4,711,845, issued Dec. 8, 1987 which is a CIP of U.S.Ser. No. 646,693 now abandoned which was a CIP of abandoned applicationSer. No. 578,133, filed Feb. 8, 1984, and assigned to the same assignee.However, any available promoter system compatible with procaryotes canbe used.

In addition to bacteria, eucaryotic microbes, such as yeast, may also beused as hosts. Laboratory strains of Saccharomyces cerevisiae, Baker'syeast, are most used although a number of other strains are commonlyavailable. While vectors employing the 2 micron origin of replicationare illustrated, Broach, J. R., Meth Enz (1983) 101: 307, other plasmidvectors suitable for yeast expression are known (see, for example,Stinchcomb, et al, Nature (1979) 282: 39, Tschempe, et al, Gene (1980)10: 157 and Clarke, L, et al, Meth Enz (1983) 101: 300). Controlsequences for yeast vectors include promoters for the synthesis ofglycolytic enzymes (Hess, et al, J Adv Enzyme Reg (1968) 7: 149;Holland, et al, Biochemistry (1978) 17: 4900). Additional promotersknown in the art include the promoter for 3-phosphoglycerate kinase(Hitzeman, et al, J Biol Chem (1980) 255: 2073), and those for otherglycolytic enzymes, such as glyceraldehyde- 3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Other promoters, which have the additional advantage oftranscription controlled by growth conditions, are the promoter regionsfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and enzymesresponsible for maltose and galactose utilization (Holland, ibid). It isalso believed terminator sequences are desirable at the 3' end of thecoding sequences. Such terminators are found in the 3' untranslatedregion following the coding sequences in yeast-derived genes. Many ofthe vectors illustrated contain control sequences derived from theenolase gene containing plasmid peno46 (Holland, M. J., et al, J BiolChem (1981) 256: 1385) or the LEU2 gene obtained from YEp13 (Broach, J.,et al, Gene (1978) 8: 121), however, any vector containing a yeastcompatible promoter, origin of replication and other control sequencesis suitable.

It is also, of course, possible to express genes encoding polypeptidesin eucaryotic host cell cultures derived from multicellular organisms.See, for example, Tissue Culture, Academic Press, Cruz and Patterson,editors (1973). Useful host cell lines include murine myelomas N51, VEROand HeLa cells, and Chinese hamster ovary (CHO) cells. Expressionvectors for such cells ordinarily include promoters and controlsequences compatible with mammalian cells such as, for example, thecommonly used early and later promoters from Simian Virus 40 (SV 40)(Fiers, et al, Nature (1978) 273: 113), or other viral promoters such asthose derived from polyoma, Adenovirus 2, bovine papilloma virus, oravian sarcoma viruses, or immunoglobulin promoters and heat shockpromoters. General aspects of mammalian cell host system transformationshave been described by Axel; U.S. Pat. No. 4,399,216 issued Aug. 16,1983. It now appears also that "enhancer" regions are important inoptimizing expression; these are, generally, sequences found upstream ofthe promoter region. Origins of replication may be obtained, if needed,from viral sources. However, integration into the chromosome is a commonmechanism for DNA replication in eucaryotes. Plant cells are also nowavailable as hosts, and control sequences compatible with plant cellssuch as the nopaline synthase promoter and polyadenylation signalsequences (Depicker, A., et al, J Mol Appl Gen (1982) 1: 561) areavailable.

C.2. Transformations

Depending on the host cell used, transformation is done using standardtechniques appropriate to such cells. The calcium treatment employingcalcium chloride, as described by Cohen, S. N., Proc Natl Acad Sci (USA)(1972) 69: 2110, is used for procaryotes or other cells which containsubstantial cell wall barriers. Infection with Agrobacterium tumefaciens(Shaw, C. H., et al, Gene (1983) 23: 315) is used for certain plantcells. For mammalian cells without such cell walls, the calciumphosphate precipitation method of Graham and van der Eb, Virology (1978)52: 546 is preferred. Transformations into yeast are carried outaccording to the method of Van Solingen, P., et al, J Bact (1977) 130:946 and Hsiao, C. L., et al, Proc Natl Acad Sci (USA) (1979) 76: 3829.

C.3. Probing mRNA by Northern Blot; Probe of cDNA or Genomic Libraries

RNA is fractionated for Northern blot by agarose slab gelelectrophoresis under fully denaturing conditions using formaldehyde(Maniatis, T., et al, Molecular Cloning (1982) Cold Spring Harbor Press,pp 202-203) or 10 mM methyl mercury (CH₃ HgOH) (Bailey, J. M., et al,Anal Biochem (1976) 70: 75-85; and Sehgal, P. B., et al, Nature (1980)288: 95-97) as the denaturant. For methyl mercury gels, 1.5% gels areprepared by melting agarose in running buffer (100 mM boric acid, 6 mMsodium borate, 10 mM sodium sulfate, 1 mM EDTA, pH 8.2), cooling to 60°C. and adding 1/100 volume of 1M CH₃ HgOH. The RNA is dissolved in 0.5×running buffer and denatured by incubation in 10 mM methyl mercury for10 min at room temperature. Glycerol (20%) and bromophenol blue (0.05%)are added for loading the samples. Samples are electrophoresed for500-600 volt-hr with recirculation of the buffer. After electrophoresis,the gel is washed for 40 min in 10 mM 2-mercaptoethanol to detoxify themethyl mercury, and Northern blots prepared by transferring the RNA fromthe gel to a membrane filter.

cDNA or genomic libraries are screened using the colony or plaquehybridization procedure. Bacterial colonies, or the plaques for phage,are lifted onto duplicate nitrocellulose filter papers (S & S typeBA-85). The plaques or colonies are lysed and DNA is fixed to the filterby sequential treatment for 5 min with 500 mM NaOH, 1.5M NaCl. Thefilters are washed twice for 5 min each time with 5×standard salinecitrate (SSC) and are air dried and baked at 80° C. for 2 hr.

The gels for Northern blot or the duplicate filters for cDNA or genomicscreening are prehybridized at 25°-42° C. for 6-8 hr with 10 ml perfilter of DNA hybridization buffer without probe (0-50% formamide,5-6×SSC, pH 7.0, 5×Denhardt's solution (polyvinylpyrrolidine, plusFicoll and bovine serum albumin; 1×=0.02% of each), 20-50 mM sodiumphosphate buffer at pH 7.0, 0.2% SDS, 20 μg/ml poly U (when probingcDNA), and 50 μg/ml denatured salmon sperm DNA). The samples are thenhybridized by incubation at the appropriate temperature for about 24-36hours using the hybridization buffer containing kinased probe (foroligomers). Longer cDNA or genomic fragment probes were labeled by nicktranslation or by primer extension.

The conditions of both prehybridization and hybridization depend on thestringency desired, and vary, for example, with probe length. Typicalconditions for relatively long (e.g., more than 30-50 nucleotide) probesemploy a temperature of 42°-55° C. and hybridization buffer containingabout 20%-50% formamide. For the lower stringencies needed foroligomeric probes of about 15 nucleotides, lower temperatures of about25°-42° C., and lower formamide concentrations (0%-20%) are employed.For longer probes, the filters may be washed, for example, four timesfor 30 minutes, each time at 40°-55° C. with 2×SSC, 0.2% SDS and 50 mMsodium phosphate buffer at pH 7, then washed twice with 0.2×SSC and 0.2%SDS, air dried, and are autoradiographed at -70° C. for 2 to 3 days.Washing conditions are somewhat less harsh for shorter probes.

C.4. Vector Construction

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

Site specific DNA cleavage is performed by treating with the suitablerestriction enzyme (or enzymes) under conditions which are generallyunderstood in the art, and the particulars of which are specified by themanufacturer of these commercially available restriction enzymes. See,e.g., New England Biolabs, Product Catalog. In general, about 1 μg ofplasmid or DNA sequence is cleaved by one unit of enzyme in about 20 μlof buffer solution; in the examples herein, typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate. Incubation times of about one hour to two hours at about 37°C. are workable, although variations can be tolerated. After eachincubation, protein is removed by extraction with phenol/chloroform, andmay be followed by ether extraction, and the nucleic acid recovered fromaqueous fractions by precipitation with ethanol. If desired, sizeseparation of the cleaved fragments may be performed by polyacrylamidegel or agarose gel electrophoresis using standard techniques. A generaldescription of size separations is found in Methods in Enzymology (1980)65: 499-560.

Restriction cleaved fragments may be blunt ended by treating with thelarge fragment of E. coli DNA polymerase I (Klenow) in the presence ofthe four deoxynucleotide triphosphates (dNTPs) using incubation times ofabout 15 to 25 min at 20° to 25° C. in 50 mM Tris pH 7.6, 50 mM NaCl, 6mM MgCl₂, 6 mM DTT and 5-10 μM dNTPs. The Klenow fragment fills in at 5'sticky ends but chews back protruding 3' single strands, even though thefour dNTPs are present. If desried, selective repair can be performed bysupplying only one of the, or selected, dNTPs within the limitationsdictated by the nature of the sticky ends. After treatment with Klenow,the mixture is extracted with phenol/chloroform and ethanolprecipitated. Treatment under appropriate conditions with S1 nucleaseresults in hydrolysis of any single-stranded portion.

Synthetic oligonucleotides may be prepared by the triester method ofMatteucci, et al (J Am Chem Soc (1981) 103: 3185-3191) or usingautomated synthesis methods. Kinasing of single strands prior toannealing or for labeling is achieved using an excess, e.g.,approximately 10 units of polynucleotide kinase to 1 nmole substrate inthe presence of 50 mM Tris, pH 7.6, 10 mM MgCl₂, 5 mM dithiothreitol,1-2 mM ATP. If kinasing is for labeling of probe, the ATP will containhigh specific activity 32ΥP.

Ligations are performed in 15-30 μl volumes under the following standardconditions and temperatures: 20 mM Tris-Cl pH 7.5, 10 mM MgCl₂, 10 mMDTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either 40 μM ATP, 0.01-0.02(Weiss) units T4 DNA ligase at 0° C. (for "sticky end" ligation) or 1 mMATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14° C. (for "blunt end"ligation). Intermolecular "sticky end" ligations are usually performedat 33-100 μg/ml total DNA concentrations (5-100 nM total endconcentration). Intermolecular blunt end ligations (usually employing a10-30 fold molar excess of linkers ) are performed at 1 μM total endsconcentration.

In the vector construction employing "vector fragments", the vectorfragment is commonly treated with bacterial alkaline phosphatase (BAP)in order to remove the 5' phosphate and prevent religation of thevector. BAP digestions are conducted at pH 8 in approximately 150 mMTris, in the presence of Na⁺ and Mg⁺² using about 1 unit of BAP per μgof vector at 60° for about one hour. In order to recover the nucleicacid fragments, the preparation is extracted with phenol/chloroform andethanol precipitated. Alternatively, religation can be prevented invectors which have been double digested by additional restriction enzymedigestion of the unwanted fragments.

C.5. Modification of DNA Sequences

For portions of vectors derived from cDNA or genomic DNA which requiresequence modifications, site specific primer directed mutagenesis isused. This technique is now standard in the art, and is conducted usinga primer synthetic oligonucleotide complementary to a single strandedphage DNA to be mutagenized except for limited mismatching, representingthe desired mutation. Briefly, the synthetic oligonucleotide is used asa primer to direct synthesis of a strand complementary to the phage, andthe resulting double-stranded DNA is transformed into a phage-supportinghost bacterium. Cultures of the transformed bacteria are plated in topagar, permitting plaque formation from single cells which harbor thephage.

Theoretically, 50% of the new plaques will contain the phage having, asa single strand, the mutated form; 50% will have the original sequence.The plaques are hybridized with kinased synthetic primer at atemperature which permits hybridization of an exact match, but at whichthe mismatches with the original strand are sufficient to preventhybridization. Plaques which hybridize with the probe are then picked,cultured, and the DNA recovered. Details of site specific mutationprocedures are described below in specific examples.

C.6. Verification of Construction

In the constructions set forth below, correct ligations for plasmidconstruction are confirmed by first transforming E. coli strain MM294,or other suitable host, with the ligation mixture. Successfultransformants are selected by ampicillin, tetracycline or otherantibiotic resistance or using other markers depending on the mode ofplasmid construction, as is understood in the art. Plasmids from thetransformants are then prepared according to the method of Clewell, D.B., et al, Proc Natl Acad Sci (USA) (1969) 62: 1159, optionallyfollowing chloramphenicol amplification (Clewell, D. B., J Bacteriol(1972) 110: 667). The isolated DNA is analyzed by restriction and/orsequenced by the dideoxy method of Sanger, F., et al, Proc Natl Acad Sci(USA) (1977) 74: 5463 as further described by Messing, et al, NucleicAcids Res (1981) 9: 309, or by the method of Maxam, et al, Methods inEnzymology (1980) 65: 499.

C.7. Hosts Exemplified

Host strains used in cloning and expression herein are as follows:

For cloning and sequencing, and for expression of construction undercontrol of most bacterial promoters, E. coli strain MM294 obtained fromE. coli Genetic Stock Center GCSC #6135, was used as the host. Forexpression under control of the P_(L) N_(RBS) promoter, E. coli strainK12 MC1000 lambda lysogen, N₇ N₅₃ cI857 SusP80, ATCC 39531 is used.

For M13 phage recombinants, E. coli strains susceptible to phageinfection, such as E. coli K12 strain DG98, are employed. The DG98strain has been deposited with ATCC July 13, 1984 and has accessionnumber 39768.

Mammalian expression has been accomplished in COS-7 and CV-1 cells.

D. Preferred Embodiments

The recombinant CSF-1 of the invention can be considered a set ofmuteins which have similar but not necessarily identical primary aminoacid sequences, all of which exhibit, or are specifically cleavable to amutein which exhibits, the activity pattern characteristic ofCSF-1--i.e. they are capable of stimulating bone marrow cells todifferentiate into monocytes, preponderantly, and, within thelimitations set forth in the Definitions section above, areimmunoreactive with antibodies raised against native CSF-1 and with thereceptors associated with CSF-1 activity. Certain embodiments of thesemuteins are, however, preferred.

The primary sequence shown in FIG. 5 for mCSF-1 has the requiredactivity, and it is, of course, among the preferred embodiments. Alsopreferred are muteins wherein certain portions of the sequence have beenaltered by either deletion of, or conservative substitution of, one ormore amino acids in mCSF-1. By a "conservative" amino acid substitutionis meant one which does not change the activity characteristics of theprotein, and in general is characterized by chemical similarity of theside chains of the two residues interchanged. For example, acidicresidues are conservatively replaced by other acidic residues, basic bybasic, hydrophobic by hydrophobic, bulky by bulky, and so forth. Thedegree of similarity required depends, of course, on the criticality ofthe amino acid for which substitution is made, and its nature. Thus, ingeneral, preferred substitutions for cysteine residues are serine andalanine; for aspartic acid residues, glutamic acid; for lysine orarginine residues, histidine; for leucine residues, isoleucine, orvaline; for trytophan residues, phenylalanine or tyrosine; and so forth.

Regions of the CSF-1 protein which are most tolerant of alterationinclude those regions of known low homology between human and mousespecies (residues 15-20 and 75-84); regions which confer susceptibilityto proteolytic cleavage (residues 51 and 52 and residues 191-193);cysteine residues not participating in disulfide linkages, or residueswhich are not absolutely essential for activity (residues 159-224). Italso appears residues 151-224 are not essential.

Therefore, particularly preferred are those CSF-1 muteins characterizedby the deletion or conservative substitution of one or more amino acidsand/or one or more sequences of amino acids between positions 159 and224 inclusive of mCSF-1 or positions 151-224 inclusive. In particular,∇₁₅₈₋ CSF-1 has CSF activity comparable to that of the native protein,even is a limited number of additional amino acid residues not relatedto CSF-1 are included at the truncated C-terminus; ∇₁₅₀₋ CSF-1 hassimilar activity. Indeed, the native protein is reported to have amolecular weight of 14-15 kd (as opposed to the 26 kd predicted from thecDNA sequence) and the hydrophobicity deduced from the recombinant(predicted) amino acid sequence corresponds to a transmembrane regionnormally susceptible to cleavage. It may, therefore, be that thetruncated version corresponds in a rough way to the CSF-1 as isolated.

Also preferred are muteins characterized by the deletion or conservativesubstitution of one or more of the amino acids at postitions 51 and 52and/or positions 191, 192 and 193 of mCSF-1. Especially preferred isgln₅₂ CSF-1; a corresponding proline substitution is not conservative,and does not yield an active CSF. Since they represent regions ofapparently low homology, another preferred set of embodiments is thatcharacterized by the deletion or conservative substitution of one ormore of the amino acids at positions 15-20 and/or positions 75-84 ofmCSF-1. Also preferred are those muteins characterized by the deletionor conservative substitution of the cysteine residue at any position notessential for disulfide bond formation. Also preferred are those muteinscharacterized by the deletion or substitution of the tyrosine residue atposition 59 of mCSF-1; particularly substitution by an aspartic acidresidue.

E. Cloning and Expression of Human CSF-1

The following illustrates the methods used in obtaining the codingsequence for human CSF-1, for disposing this sequence in expressionvectors, and for obtaining expression of the desired protein.

E.1. Purification of Native Human CSF-1 and Probe Design

Human urinary CSF-1 was partially purified by standard methods asdescribed by Das, S. K., et al, Blood (1981) 58: 630, followed by anaffinity purification step using a rat monoclonal antibody to murineCSF-1, designated YYG106, attached to a Sepharose B column (Stanley, E.R., Methods Enzymol (1985) 116: 564). The final step in purification wasreverse phase HPLC in a 0.1% TFA/30% acetonitrile--0.1% TFA/60%acetonitrile buffer system.

For MIAPaCa CSF-1, which was produced serum-free by induction withphorbol myristic acetate, the cell supernatant was subjected to calciumphosphate gel chromatography (according to Das (supra)), followed byaffinity chromatography using lentil lectin (in place of the ConAaffinity step of Das), and then to the immunoaffinity step employing theYYG106 monoclonal antibody conjugated to Sepharose B and to the reversephase HPLC, both as above described.

The urinary and MIAPaCa proteins, having been purified to homogeneity,were subjected to amino acid sequencing using Edman degradation on anautomated sequencer. Sufficient N-terminal sequence of human CSF wasdetermined to permit construction of probes shown in FIG. 3.

E.2. Preparation of the Human Genomic Sequence

A human genomic sequence encoding CSF-1 was obtained from the Maniatishuman genomic library in λ phage Charon 4 using probes designed toencode the N-terminal sequence of human protein. The library wasconstructed using partial HaeIII/AluI digestion of the human genome,ligatiion to EcoRI linkers, and insertion of the fragments into EcoRIdigested Charon 4 phage. A Charon 4A phage containing the CSF-1 sequenceas judged by hybridization to probe as described below, and designatedpHCSF-1, was deposited with the American Type Culture Collection (ATCC)on Apr. 2, 1985 and has accession no. 40177. Upon later study of thisphage, it was found that rearrangements and/or deletions had occurredand the correct sequences were not maintained. Therefore, an alternativecolony obtained from the genomic library in identical fashion, andpropogated to confirm stability through replication, was designatedpHCSF-1a and was deposited with ATCC on May 21 1985, and given accessionnumber 40185. pHCSF-1a contained an 18 kb insert and was capable ofgenerating restriction enzyme digests which also hybridized to probe,and was used for sequence determination and additional probeconstruction as outlined below.

If the CSF-1 encoding sequence is present in its entirety its presencecan be demonstrated by expression in COS-7 cells, as described byGluzman, Y., Cell (1981) 23: 175. The test fragment is cloned into aplasmid derived from pBR322 which has been modified to contain the SV40origin of replication (pGRI Ringold, G., J Mol Appl Genet (1982) 1:165-175). The resulting high copy number vectors are transformed intoCOS-7 cells and expression of the CSF-1 gene assayed after 24, 48, and72 hours by the radioreceptor assay method described by Das (supra).Expression is under control of the native CSF-1 control sequences. TheHindIII digests of the approximately 18 kb insert of pHCSF-1a tested inthis manner failed to express, thus indicating that HindIII digests intothe gene. This was confirmed by subsequent mapping.

However, for initial sequencing, a 3.9 kb HindIII fragment was obtainedfrom the pHCSF-1a phage and cloned into M13 cloning vectors.

The HindIII fragment has been partially sequenced, and the results areshown in FIG. 4, along with a deduced peptide sequence. It contains thecorrect codons for the portion of the human CSF-1 protein for which theamino acid sequence had been determined, as set forth in FIG. 1. Thepresence of an intron of approximately 1400 bp was deduced from theavailable amino acid sequence. In addition, based on the genomicsequence encoding amino acids 24-34 (see overlined portion of FIGS. 4and 5), a 32-mer probe for the cDNA library was constructed and employedas described below.

In more detail, to obtain the genomic clone, pHCSF-1a, the Maniatislibrary was probed using two mixtures of oligomers shown in FIG. 3. EK14and EK15 were selected, although the other oligomers shown are useful aswell. A "full length" probe for the N-terminal sequence, EK14, was usedas a mixture of sixteen 35-mers. A shorter oligomer, EK15, was employedas a mixture of sixty-four 18-mers. Phage hybridizing to both kinasedprobes were picked and cultured by infection of E. coli DG98 or othercompetent strain.

Specific conditions for probing with EK14 and EK15 are as follows; forEK14, the buffer contained 15% formamide, 6×SSC, pH 7.0, 5× Denhardt's,20 mM sodium phosphate, 0.2% SDS and 50 μg/ml denatured salmon spermDNA. Prehybridization and hybridization were conducted at 42° C. and thefilters were washed in 2×SSC at 52° C. For EK15, similar conditins wereused for hybridization and prehybridization except for the formamideconcentration, which was 0%; washing was at a slightly lowertemperature, 42° C.

The approximately 18 kb DNA insert isolated from the positivelyhybridizing phage pHCSF-1a was treated with HindIII and the fragmentswere subjected to electrophoresis on agarose gel according to the methodof Southern. The gels were replicated onto nitrocellulose filters andthe filters were probed again with EK14 and EK15. Both probes hybridizedto a 3.9 kb fragment.

The positive fragment was excised from the gel, eluted, and subclonedinto HindIII-treated M13mp19 for dideoxy sequencing. A partial sequenceis shown in FIG. 4. The underlining corresponds precisely to thepreviously determined N-terminal sequence of human CSF-1; the residueswith dot subscripts are homologous to the murine sequence.

In FIG. 4, the 1.4 kb intron region between the codons for amino acids22 and 23, as deduced from the human sequence determined from thepurified protein, is shown untranslated. The sequence upstream of theN-terminal residues contains the putative leader; the translation of theportion of this leader immediately adjacent to the mature protein, whichwas tentatively verified by the preliminary results of sequencing of thecDNA clone (see below) is shown. The upstream portions are, however, notshown translated; these portions are confirmed by comparison to the cDNAto comprise an intron.

Further sequencing to obtain about 13 kb of the entire 18 kb gene showsthat the gene contains 9 exons separated by 8 introns. The regions ofthe mature protein cDNA correspond exactly to the genomic exon codonsexcept for codon 59, as further described below.

An additional M13 subclone was obtained by digestion of the HindIII 3.9kb fragment with PstI to generate a 1 kb PstI/PstI fragment whichincludes the known N-terminal sequence and about 1 kb of additionalupstream sequence.

E.3. cDNA Encoding Human CSF-1

psCSF-17

The human derived pancreatic carcinoma cell line MIAPaCa-2 was used as asource of mRNA to validate probes and for the formation of a cDNAlibrary containing an intronless form of the human CSF-1 codingsequence. The MIApaCa cell line produces CSF-1 at a level approximately10 fold below that of the murine L-929 cells.

Negative control mRNA was prepared from MIAPaCa cells maintained inserum-free medium, i.e. under conditions wherein they do not produceCSF-1. Cells producing CSF-1 were obtained by reinducing CSF-1production after removal of the serum.

Cells were grown to confluence in roller bottles using Dulbecco'sModified Eagles' Medium (DMEM) containing 10% fetal calf serum, andproduce CSF-1 at 2000-6000 units/ml. The cell cultures were washed, andreincubated serum-free to suppress CSF-1 formation. For negativecontrols, no detectable CSF-1 was produced after a day or two. Reinducedcells were obtained by addition of phorbol myristic acetate (100 ng/ml)to obtain production after several days of 1000-2000 units/ml.

The mRNA was isolated by lysis of the cell in isotonic buffer with 0.5%NP-40 in the presence of ribonucleoside vanadyl complex (Berger, S. L.,et al, Biochemistry (1979) 18: 5143) followed by phenol chloroformextraction, ethanol precipitation, and oligo dT chromatography, and anenriched mRNA preparation obtained. In more detail, cells are washedtwice in PBS (phosphate buffered saline) and are resusupended in IHB(140 mM NaCl, 10 mM Tris, 1.5 mM MgCl₂, pH 8) containing 10 mM vanadyladenosine complex (Berger, S. L., et al, supra).

A non-ionic detergent of the ethylene oxide polymer type (NP-40) isadded to 0.5% to lyse the cellular, but not nuclear membranes. Nucleiare removed by centrifugation at 1,000×g for 10 min. The post-nuclearsupernatant is added to two volumes of TE (10 mM Tris, 1 mMethylenediaminetetraacetic acid (EDTA), pH 7.5) saturated phenolchloroform (1:1) and adjusted to 0.5% sodium dodecyl sulfate (SDS) and10 mM EDTA. The supernatant is re-extracted 4 times and phase separatedby centrifugation at 2,000×g for 10 min. The RNA is precipitated byadjusting the sample to 0.25M NaCl, adding 2 volumes of 100% ethanol andstoring at -20° C. The RNA is pelleted at 5,000×g for 30 min, is washedwith 70% and 100% ethanol, and is then dried. Polyadenylated (poly A⁺)messenger RNA (mRNA) is obtained from the total cytoplasmic RNA bychromatography on oligo dT cellulose (Aviv, J., et al, Proc Natl AcadSci (1972) 69: 1408-1412). The RNA is dissolved in ETS (10 mM Tris, 1 mMEDTA, 0.5% SDS, pH 7.5) at a concentration of 2 mg/ml. This solution isheated to 65° C. for 5 min, then quickly chilled to 4° C. After bringingthe RNA solution to room temperature, it is adjusted to 0.4 M NaCl andis slowly passed through an oligo dT cellulose column previouslyequilibrated with binding buffer (500 mM NaCl, 10 mM Tris, 1 mM EDTA, pH7.5 0.05% SDS). The flow-through is passed over the column twice more.The column is then washed with 10 volumes of binding buffer. Poly A⁺mRNA is eluted with aliquots of ETS, extracted once with TE-saturatedphenol chloroform and is precipitated by the addition of NaCl to 0.2Mand 2 volumes of 100% ethanol. The RNA is reprecipitated twice, iswashed once in 70% and then in 100% ethanol prior to drying.

Total mRNA was subjected to 5-20% by weight sucrose gradientcentrifugation in 10 mM Tris HCl, pH 7.4 1 mM EDTA, and 0.5% SDS using aBeckman SW40 rotor at 20° C. and 27,000 rpm for 17 hr. The mRNAfractions were then recovered from the gradient by ethanolprecipitation, and injected into Xenopus oocytes in the standardtranslation assay. The oocyte products of the RNA fractions were assayedin the bone marrow proliferation assay (as described by Moore, R. N., etal, J Immunol (1983) 131: 2374, and of Prystowsky, M. B., et al, Am JPathol (1984) 114: 149) and the fractions themselves were assayed by dotblot hybridization to a 32-mer probe corresponding to the DNA in thesecond exon of the genomic sequence (exon II probe). (The overlining inFIGS. 4 and 5 shows the exon II probe.) These results are summarized inFIG. 7.

The broken line in FIG. 7A shows the respose in the bone marrowproliferation assay of the supernatants from the Xenopus oocytes; FIG.7B shows the dot-blot results. The most strongly hybridizing fraction,11, corresponds to a size slightly larger than the 18S marker, while themost active fractions 8 and 9 correspond to 14-16S. Fractions 8, 9 and11 were used to form an enriched cDNA library as described below.

(The mRNA was also fractionated on a denaturing formaldehyde gel,transferred to nitrocellulose, and probed with exon II probe. Severaldistinct species ranging in size from 1.5 kb to 4.5 kb were found, evenunder stringent hybridization conditions. To eliminate the possibilityof multiple genes encoding CSF-1, digests of genomic DNA with variousrestriction enzymes were subjected to Southern blot and probed usingpcCSF-17 DNA. The restriction pattern was consistent with the presenceof only one gene encoding CSF-1.)

The enriched mRNA pool was prepared by combining the mRNA from thegradient fractions (8 and 9) having the highest bone marrowproliferative activity, although their ability to hybridize to probe isrelatively low (14S-16S) with the fractions (11) hybridizing mostintensely to probe (slightly larger than 18S). Higher molecular weightfractions which also hybridized to exon II probe were not includedbecause corresponding mRNA from uninduced MIAPaCa Cells also hybridizedto exonII probe.

cDNA libraries were prepared from total or enriched human mRNA in twoways. One method uses λgt10 phage vectors and is described by Huynh, T.V., et al, in DNA Cloning Techniques: A Practical Approach IRL Press,Oxford 1984, D. Glover, Ed.

A preferred method uses oligo dT priming of the poly A tails and AMVreverse transcriptase employing the method of Okayama, H., et al, MolCell Biol (1983) 3: 280-289, incorporated herein by reference. Thismethod results in a higher proportion of full length clones than doespoly dG tailing and effectively uses as host vector portions of twovectors therein described, and readily obtainable from the authors,pcDVl and pLl. The resulting vectors contain the insert between vectorfragments containing proximal BamHI and XhoI restriction sites; thevector contains the pBR322 origin of replication, and Amp resistancegene and SV40 control elemetns which result in the ability of the vectorto effect expression of the inserted sequences in COS-7 cells.

A 300,000 clone library obtained from above enriched MIAPaCa mRNA by theOkayama and Berg method was then probed under conditions of highstringency, using the exon II probe. Ten colonies hybridizing to theprobe were picked and colony purified. These clones were assayed for thepresence of CSF-1 encoding sequences by transient expression in COS-7cells. The cloning vector, which contains the SV40 promoter, was usedper se in the transformation of COS-7 cells.

Plasmid DNA was purified from the 10 positive clones using a CsClgradient, and the COS-7 cells transfected using a modification (Wang, A.M., et al, Science (1985) 228: 149) of the calcium phosphatecoprecipitation technique. After incubation for three days, CSF-1production was assayed by subjecting the culture supernatants to theradioreceptor assay performed substantially as disclosed by Das, S. K.,et al, Blood (1981) 58: 630, and to a colony stimulation (bone marrowproliferation) assay performed substantially as disclosed by Prystowsky,M. B., et al, Am J Pathol (1984) 114: 149. Nine of the ten clones pickedfailed to show transient CSF-1 production in COS-7 cells. One clone,which did show expression, was cultured, the plasmid DNA isolated, andthe insert was sequenced. The DNA sequence, along with the deduced aminoacid sequence, are shown in FIG. 5. The full length cDNA is 1.64 kb andencodes a mature CSF-1 protein of 224 amino acids. The clone wasdesignated CSF-17 with Cetus depository number CMCC 2347 and wasdeposited with the American Type Culture Collection on June 14 1985, asaccession no. 53149. The plasmid bearing the CSF-1 encoding DNA wasdesignated pcCSF-17.

Mutein-Encoding Sequences

Modifications were made of the pcCSF-17 inserts to provide correspondingplasmids encoding muteins of the mCSF-1 protein. For site-specificmutagenesis, pcCSF-17 and M13mp18 were digested with the samerestriction enzyme excising the appropriate region of the CSF-1 codingsequence, and the excised sequence ligated into the M13 vector. Secondstrand synthesis and recovery of the desired mutated DNA used thefollowing oligonucleotide primers:

for pro₅₂ CSF-1, 5'-TACCTTAAACCGGCATTTCTC-3', which creates a new HpaIIsite at codons 52-53;

for gln₅₂ CSF-1, 5'-TACCTTAAACAGGCCTTTCTC-3', which creates a new StuIsite at codons 52-53;

for asp₅₉ CSF-1, 5'-GGTACAAGATATCATGGAG-3', which creates a new EcoRVsite at codons 59-60.

After second strand extension using Klenow, the phage were transformedinto E coli DG98 and the resulting plaques screened with kinased labeledprobe. After plaque purification, the desired mutated inserts werereturned to replace the unmutated inserts in pcCSF-1, yieldingpCSF-pro52, pCSF-gln52, and pCSF-asp59, respectively.

Plasmids containing three deletion mutants which encode ∇₁₅₈₋ CSF-1 werealso prepared: pCSF-Bam, pCSF-BamBcl, and pCSF-BamTGA. For pCSF-Bam,pcCSF-17 was digested with BamHi, and the upstream BamHI/BamHI fragmentof the coding region was isolated and religated to the vector fragment.The ligation mixture was transformed into E coli MM294 and plasmids withthe correct orientation isolated. The resulting pCSF-Bam encodes 158amino acids of the CSF-1 protein fused to six residues derived from thevector at the C-terminus: arg-his-asp-lys-ile-his.

For pCSF-BamBcl, which contains the entire CSF-1 encoding sequence,except that the serine at position 159 is mutated to a stop codon, thecoding sequence was excised from pcCSF-17 and ligated into M13 forsite-specific mutagenesis using the primer:5'-GAGGGATCCTGATCACCGCAGCTCC-3'. This results in a new BclI site atcodons 159-160. The mutated DNA was excised with BstXI/EcoRI and ligatedinto the BstXI/EcoRI digested pcCSF-17, the ligation mixture wastransformed into E coli DG105, a dam host, and the plasmid DNA isolated.

For pCSF-BamTGA, in which the codons downstream of the 159-stop aredeleted, pCSF-BamBcl was digested with XhoI and BcII, and the insertligated into XhoI/BamHI digested pcCSF-17.

In addition, pCSF-Gly150, which contains a TGA stop codon instead ofhistidine at position 151, was prepared from the pcCSF-17 insert bysite-specific mutagenesis using the appropriate primer, as describedabove.

E.4. Transient Expression of CSF-1

Expression of pcCSF-17

The expression of plasmid DNA from CSF-17 (pcCSF-17) in COS-7 cells wasconfirmed and quantitated using the bone marrow proliferation assay, thecolony stimulation assay and the radioreceptor assay. It will berecalled that the specificity of the bone marrow proliferation assay forCSF-1 resides only in the ability of CSF-1 antiserum to diminishactivity; that for the colony stimulation assay, in the nature of thecolonies obtained. Both assays showed CSF-1 production to be of theorder of several thousand units per ml.

Bone Marrow Proliferation

For the bone marrow stimulation assay, which measures biologicalactivity of the protein, bone marrow cells from Balb/C mice were treatedwith serial dilutions of the 72 hour supernatants and proliferation ofthe cells was measured by uptake of labeled thymidine, essentially asdescribed by Moore, R. N., et al, J Immunol (1983) 131: 2374;Prystowsky, M. B., et al, Am J Pathol (1984) 114: 149. The medium frominduced MIAPaCa cells was used as control. Specificity for CSF-1 wasconfirmed by the ability of rabbit antisera raised against human urinaryCSF-1 to suppress thymidine uptake. The results for COS-7 cellsupernatants transfected with pcCSF-17 (CSF-17 supernatant) at a 1:16dilution are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                    .sup.3 H-thymidine incorporation (cpm)                                        no      normal  antihuman                                                     add'ns  serum   CSF-1 serum                                       ______________________________________                                        medium         861       786    2682                                          MIAPaCa supernate                                                                           12255     16498   3302                                          CSF-17 supernate                                                                            16685     21996   2324                                          ______________________________________                                    

(The antihuman CSF-1 serum was prepared as described by Das, et al,supra.)

The MIAPaCa supernatant (at the 1:16 dilution used above) contained 125U/ml CSF activity corresponding to 2000 U/ml in the undilutedsupernatant, where 1 unit of colony stimulating activity is defined asthe amount of CSF needed to produce one colony from 10⁵ bone marrowcells/ml in the assay of Stanley, E. R., et al, J Lab Clin Med (1972)79: 657.

These data show that the bone marrow stimulating activity is associatedwith CSF-1, since thymidine uptake is inhibited by anti-CSF-1 serum.Regression of results in this bone marrow proliferation assay obtainedat four dilutions ranging from 1:8 to 1:64 gave an estimated activityfor CSF-1 in CSF-17 supernatants of 2358 U/ml, which was diminished to424 U/ml in the presence of antiserum, but showed an apparent increaseto 3693 U/ml in the presence of non-immune serum. This was comparable tothe levels shown in the radioreceptor assay below.

Colony Stimulation

Direct assay of the CSF-17 supernatants for colony stimulation (Stanley,E. R., et al, J Lab Clin Med (supra)) showed 4287 U/ml, which wassubstantially unaffected by the presence of non-immune serum but reducedto 0 U/ml in the presence of rabbit antihuman CSF-1. This compares to2562 U/ml in the MIAPaCa supernatants. Eighty-five percent of thepcCSF-17 transformed COS-7 supernatant induced colonies had mononuclearmorphology; MIAPaCa supernatant induced colonies showed a 94%macrophage-6% granulocyte ratio.

Radioreceptor Assay

The radioreceptor assay measures competition between ¹²⁵ I-labeled CSF-1and the test compound for specific receptors on J774.2 mouse macrophagecells. MIAPaCa supernatant, assayed for colony stimulating activity asabove, was used as a standard (2000 U/ml). The CSF-1 concentration ofthe pcCSF-17 transformed COS-7 supernatant was found to be 2470 U/mlbased on a 1:10 dilution and 3239 U/ml based on a 1:5 dilution.

Thus, comparable values for CSF-1 concentration in the media of COS-7cells transformed with pcCSF-17 were found in all assays.

Expression of Muteins

In a similar manner to that described above for pcCSF-17, themutein-encoding plasmids were transfected into COS-A2 cells andtransient expression of CSF-1 activity assayed by the bone marrowproliferation assay and by radioimmunoassay using anti-CSF antibodies.The expression product of pCSF-pro52 was inactive, indicating that, asexpected, substitution by proline is not conservative. All other muteinsshowed activity in both assays as shown by the results below:

    ______________________________________                                        Expression of CSF-1 Constructs in COS Cells                                            Radio-    Bone Marrow Assay                                          CSF-1      immunoassay Proliferation                                                                             Colony                                     Plasmid    (units/ml)  (units/ml)  (units/ml)                                 ______________________________________                                        pcCSF-17   3130        2798        11,100                                                3080        3487        9750                                                  3540        3334        11,500                                     pCSF-pro52 54.8        <25         <100                                                  51.9        <25         <100                                                  45.3        <25         <100                                       pCSF-gln52 1890        2969        6200                                                  2250        2308        5500                                                  1910        2229        4400                                       pCSF-asp59 3210        3381        9000                                                  4680        3417        6800                                                  3470        2812        10,600                                     pCSF-Bam   9600        8048        22,600                                                8750        8441        21,900                                                8400        10,995      21,700                                     pCSF-BamBcl                                                                              8800                    26,000                                                10,700                  21,600                                                15,450                  24,200                                     pCSF-BamTGA                                                                              8450                    22,600                                                7550                    23,200                                                9700                    20,000                                     pCSF-Gly150                                                                              26,850                  55,710                                     ______________________________________                                    

E.5. Stable Expression of CSF-1

The COS-7 system provides recombinant CSF-1 by permitting replication ofand expression from the vector sequences. It is a transient expressionsystem.

The human CSF-1 sequence can also be stably expressed in procaryotic oreucaryotic systems. In general, procaryotic hosts offer ease ofproduction, while eucaryotes permit the use of the native signalsequence and carry out desired post-translational processing. This maybe especially important in the case of CSF-1 since the native protein isa dimer. Bacteria produce CSF-1 as a monomer, which would then besubjected to dimerizing conditions after extraction.

Procaryotic Expression

For procaryotic expression, the cDNA clone, or the genomic sequence withintrons excised by, for example, site-specific mutagenesis, is alteredto place an ATG start codon immediately upstream of the glutamic acid atthe N-terminus, and a HindIII site immediately upstream of the ATG inorder to provide a convenient site for insertion into the standard hostexpression vectors below. This can be done directly using insertionsite-specific mutagenesis with a synthetic oligomer containing a newsequence complementary to the desired AAGCTTATG, flanked by nucleotidesequences complementary to the native leader and N-terminal codingsequences.

For cDNA obtained using the method of Okayama and Berg, the DNA fragmentcontaining the entire coding sequence is excised from pcCSF-17 or thecorresponding mutein vector by digestion with XhoI (at sites retainedfrom the host cloning vector), isolated by agarose gel electrophoresis,and recovered by electroelution. To carry out the mutagenesis, the hostbacteriophage M13mp18 DNA is also treated with SalI and ligated with thepurified fragment under standard conditions and transfected into frozencompetent E. coli K12 strain DG98. The cells are plated on mediacontaining 5×10⁻⁴ M isopropyl thiogalactoside (IPTG) obtained from SigmaChem. (St. Louis, MO) and 40 μg/ml X-gal. Non-complementing whiteplaques are picked into fresh media. Mini-cultures are screened forrecombinant single strand phage DMA of the expected size, and thestructure of the desired recombinant phage is confirmed usingrestriction analysis.

A 34-mer complementary to the N-terminal and leader encoding portions ofthe CSF-1 sequence, but containing the complement to the desiredAAGCTTATG sequence is synthesized and purified according to theprocedures set forth in C.4. A portion of this 34-mer preparation isradiolabeled according to a modification of the technique of Maxam andGilbert (Maxam, A., et al, Methods in Enzymology (1980) 68: 521,Academic Press) as set forth in C.4 above.

To perform the mutagenesis the above prepared recombinant bacteriophageis prepared in E. coli K12 strain DG98 and the single strand phage DNApurified. One pmole of single strand phage DNA and 10 pmoles of theabove synthetic nucleotide primer (not kinased) are annealed by heatingfor 1 min at 67° C., and then 30 min at 37° C. in 15 μl 20 mM Tris-Cl,pH 8, 20 mM MgCl₂, 100 mM NaCl, 20 mM 2-mercaptoethanol. The annealedDNA is incubated with DNA polymerase I (Klenow) and 500 μM dNTPs for 30min, 0° C. and then brought to 37° C. Aliquots (0.05 or 0.25 pmole) areremoved after 5 min, 20 min, and 45 min, transformed into E. coli K12strain DG98 and plated.

After growth, the plates are chilled at 4° C. and plaques lifted withPalI membranes obtained from Biodyne of S&S filters (1-2 min in thefirst filter, more than 10 min for the second filter). The filters aredenatured in 2.5M NaCl, 0.5M NaOH (5 min). The denaturing medium isneutralized with 3M sodium acetate to pH 5.5, or with 1M Tris-Cl, pH 7.5containing 1M NaCl, the filters baked at 80° C. in vacuo for 1 hr, andthen prehybridized at high stringency. The filters are then probed withthe kinased synthetic 34-mer prepared above at high stringency, washed,and autoradiographed overnight at -70° C.

The RF form of the desired mutated phage is treated with EcoRI, bluntedwith Klenow, and then digested with HindIII to excise the gene as aHindIII/blunt fragment. (In a strictly analogous manner, the CSF-1encoding sequence from pMCSF may be obtained and modified.)

This fragment containing the human (or murine) CSF-1 encoding sequenceis then ligated with HindIII/BamHI (blunt) digested pPLOP or pTRP3 (seebelow) to place the coding sequence containing the ATG start codonimmediately downstream from the P_(L) or trp promoter respectively.These resulting plasmids are transformed into E. coli MC1000 lambdalysogen or MM294, and the cells grown under non-inducing conditions andthen induced by means appropriate to the promoter. The cells areharvested by centrifugation, sonicated and the liberated CSF-1solubilized. The presence of human (or murine) CSF-1 is confirmed bysubjecting the sonicate to the colony stimulating assay set forth above.

In addition, the plasmid pFC54.t (ATCC 39789) which contains the P_(L)promoter and the Bacillis thuringiensis positive retroregulatorysequence (as described in EPO Application Publication No. 717,331,published Mar. 29, 1985) was used as a host vector. pFC54.5 was digestedwith HindIII/BamHI(blunt), and the desired coding sequences ligated intothe vector using the HindIII/EcoRI(blunt) excised fragment from pcCSF-17or the mutein encoding vectors described above. After transformationinto E. coli MC1000 lambda lysogen, and induction, CSF-1 production wasobtained and verified as described above.

Finally, it was possible to improve the level of CSF-1 production fromthe foregoing constructs by altering the third nucleotide in each of thefirst six codons of the N-terminus. pFC54.5 containing the CSF-1encoding fragment was digested with HindIII/BstXI, and the excisedfragment (which contains the ATG and a short portion of the subsequentcoding sequence) was replaced by a synthetic HindIII/BstXI segmentwherein the first six codons have the sequence: GAAGAAGTTTCTGAATAT. Theresulting analogous expression vector represents no change in the aminoacid sequence encoded; however, the levels of expression are improvedwhen this modified vector is used.

Eucaryotic Expression

The Okayama-Berg plasmid pcCSF-17, containing the cDNA encoding humanCSF-1 under control of the SV40 promoter, can also be used to effectstable expression in monkey CV-1 cells, the parent cell line from whichthe COS-7 line was derived. The corresponding vectors encoding themuteins as described above can also be used in an exactly analogous way.The host monkey CV-1 cells were grown to confluence and thencotransformed using 10 μg pcCSF-17 and various amounts (1, 2, 5 and 10μg) of PRSV-NEO2 (Gorman, C., et al, Science (1983) 221: 551-553) per500,000 cells. The transformants were grown in DMEM with 10% FBS mediumcontaining 100 μg/ml of G418 antibiotic, to which the pRSV-NEO2 plasmidconfers resistance. The CV-1 cell line showed a G418 transformationfrequency of 10⁻⁵.12 colonies per 10⁶ cells per μg DNA.

The CV-1 cells were cotransformed as described above and selected inG418-containing medium. Resistant clones were tested for stability ofthe G418-resistant phenotype by growth in G418-free medium and thenreturned to G418-containing medium. The ability of these cultures tosurvive when returned to antibiotic-containing medium suggests that thepRSV-NEO2 DNA was integrated permanently into the cell genome. Sincecells stably transformed with a marker plasmid have about 50%probability of having integrated the DNA of a cotransfecting plasmid,about half of these cells will also contain pcCSF-17 DNA in theirchromosomal DNA.

Several clones of the C418-resistant pools of CV-1 cells which weredemonstrated to be stably transformed as above were picked and grown induplicate flasks to near confluence. One flask of each duplicate wasinfected with SV-40 virus at a multiplicity of infection of 5, and themedium was harvested 6 days after infection for assay for CSF-1 using aradioimmunoassay. The immunoassay is based on competition of ¹²⁵I-labeled MIAPaCa CSF-1 for "Rabbit 52" polyclonal antiserum raisedagainst purified human urinary CSF-1.

One of the selected CV-1 clones showed 2335 U/ml production of CSF-1,according to this assay, whereas cells not infected with SV-40 showedless than 20 U/ml. Controls using COS-7 cells transformed with 10 μgpcCSF-17 showed 2400 U/ml CSF-1 production without SV-40 infection.

The CSF-1 producing CV-1 cell line contains the pcCSF-17 DNA stablyintegrated into its genome, and thus can be used for stable productionof CSF-1 upon infection with SV-40. Infection is presumed to "rescue"the pcCSF-17 DNA from the genome, and provide the SV-40 T-antigennecessary for replication of the rescued DNA. Without SV-40 infection,the integrated pcCSF-17 DNA is not effectively expressed.

Optimization of the expression of the CSF-1 encoding sequence by theCV-1 (CSF-17) cell line showed 6500-8000 U/ml when measured by theradioimmunoassay six days after SV-40 infection using a multiplicity ofinfection of at least 1, and a 10% FBS medium. Studies on expressionlevels at a multiplicity of 10 showed comparable production, butproduction was reduced upon removal of the FBS from the medium on thesecond day after infection.

In the alternative, appropriate control systems and host vectorspermitting expression in eucaryotic hosts may be used to receive theCSF-1 encoding inserts. For example, CHO cells and suitable vectors maybe used, as described in U.S. Ser. No. 438,991, filed Nov. 1, 1982, nowadandoned assigned to the same assignee and incorporated herein byreference.

E.6. Activity of CSF-1

Additional definition of the activity of CSF-1 was provided usingpartially purified MIAPaCa CSF-1 or murine L cell CSF-1 as models forthe CV-1-produced recombinant material. CSF-1 was shown to enhance theproduction of interferon and tumor necrosis factor (TNF) by inducedhuman monocytes by up to 10-fold. CSF-1 also was demonstrated tostimulate macrophage antitumor toxicity.

Stimulation of TNF Production by Human Monocytes

MIAPaCa CSF-1 was purified from the supernatant by calcium phosphate gelfiltration and lentil lectin chromatography. For assay of lymphokineproduction, peripheral blood-adherent cells were incubated in duplicateflasks containing 10⁷ cells each. One flask was treated with 1000 U/mlCSF-1 purified as above. After 3 days, the cells were harvested, andwashed, and resuspended at a cell concentration of 5×10⁵ /ml and platedin 24-well plates at 0.5 ml/well. The wells were treated with 10 μg/mlLPS and 20 ng/ml PMA for 48 hr and the supernatants were harvested forTNF assay. Cells treated with CSF showed TNF secretions approximatelynine-fold higher than the untreated cells (1500 U/ml, compared to 162U/ml).

Stimulation of Interferon Production by Human Monocytes

In an analogous experiment to determine the effect of CSF-1 oninterferon production, peripheral blood-adherent cells were incubatedfor 3 days in the presence and absence of 1000 U/ml CSF-1, as describedabove, harvested, resuspended at 5×10⁵ /ml, and plated in a 25-wellplate, as described above. The cells were induced for interferonproduction by addition of varying amounts of poly(I): poly(C). Thesupernatants were assayed for interferon production by their cytopathiceffect on VSV-infected GM 2504 cells. The CSF-1-stimulated cells showedproduction of 100 U/ml when induced with 50 μg/ml poly(I): poly(C), asdescribed by McCormick, F., et al, Mol Cell Biol (1984) 4: 166, whereascomparably induced untreated cells produced less than 3 U/ml.

Stimulation of Myeloid CSF Production by Human Monocytes

Monocytes were incubated ±CSF-1 for 3 days and then induced forproduction of myeloid CSF as in Table 1. The three representativeexperiments shown used blood from different donors.

                  TABLE 2                                                         ______________________________________                                        Myeloid CSF (U/ml)                                                                   Exp. 1    Exp. 2      Exp. 3                                           Induction                                                                              -CSF    +CSF    -CSF  +CSF  -CSF  +CSF                               ______________________________________                                        medium   0       0       0     0     0     0                                  0.1 μg/ml                                                                           --      --      0     0     0     80 ±                            LPS                                        17                                 1 μg/ml LPS                                                                         0       700 ±                                                                              40 ±                                                                             200 ±                                                                            103 ±                                                                            377 ±                                            72      20    20    12    57                                 0.1 μg/ml                                                                           --      --      617 ±                                                                            993 ±                                                                            1120 ±                                                                           1280 ±                          LPS + 2                  50    101   82    60                                 ng/ml PMA                                                                     1 μg/ml LPS                                                                         283 ±                                                                              983 ±                                                                              360 ±                                                                            1400 ±                                                                           537 ±                                                                            1080 ±                          + 2 ng/ml                                                                              42      252     92    180   47    12                                 PMA                                                                           2 ng/ml  --      370 ±                                                                              297 ±                                                                            183 ±                                                                            380 ±                                                                            716 ±                           PMA              17      6     15    52    76                                 ______________________________________                                    

Therefore, CSF-1 stimulates myeloid CSF production.

Stimultion of Tumor Cell Killing by Murine Macrophage; Comparison toother Colony Stimulating Factors

To assay macrophage stimulation, murine CSF-1 obtained fromL-cell-conditioned medium, was used as a model for the recombinantlyproduced CSF-1 from pcCSF-17 in an assay which showed stimulation of theability of murine macrophages to kill sarcoma targets. In this assay,normal 2 hr adherent C3H/HeN mouse peritoneal macrophages were incubatedfor 1 day in vitro with and without CSF-1 and then mixed at a 20:1 ratiowith ³ H-thymidine-labeled mouse sarcoma TU5 cells along with 10% v/vconA-induced (10 μg/ml) spleen lymphokine (LK), which contains gammainterferon. The release of labeled thymidine over the following 48 hrwas used as a measure of tumor cell killing. The effect of adding CSF-1as murine L-cell-conditioned medium containing 1200 U/ml CSF-1 is shownin the following table.

    ______________________________________                                        Treatment                 Increase Due                                        DAY     DAY            Kill   to CSF-1                                        0→1                                                                            1→3     %      %                                               ______________________________________                                        --      --             13                                                     --      LK             39                                                     --      CSF-1 + LK     49     26                                              CSF-1   LK             51     31                                              CSF-1   CSF-1 + LK     60     54                                              --      --              3                                                     --      LK             35                                                     --      CSF-1 + LK     47     34                                              CSF-1   --              7                                                     CSF-1   LK             49     40                                              CSF-1   CSF-1 + LK     69     97                                              ______________________________________                                    

Increase in the ability to kill the target cells was noted whether CSF-1was added during the preliminary 1 day of growth or during the period ofinduction; however, the most dramatic effects were observed with CSF-1was present during both of these periods.

The possibility of contaminating bacterial lipopolysaccharide (LPS) asthe cause of stimulation of monocytes and macrophages was excluded: TheLPS content of the applied CSF-1 was low (21 0.3 ng/3000 U CSF-1, byLimulus amoebocyte lysate assay); activity was removed by application toan anti-CSF-1 column; polymyxin B was used to neturalize LPS; themacrophages from C3H/HeJ mice respond to CSF-1 but not to LPS.

CSF-GM was prepared from 6 mouse lungs obtained 5 hours after IVadministration of 5 μg LPS. The lungs were chopped and incubated for 3days in serum free medium, and the supernatant was depleted of CSF-1using a YYG106 affinity column (CSF-1 content reduced from 270 U/ml to78 U/ml). CSF-G was prepared from similarly treated LDI serum freemedium. Both CSF-GM and CSF-G contents were assayed at 2000 U/ml bycolony stimulating assay.

The peritoneal macrophages were incubated with 40% of either of theforegoing media or with L-cell medium assayed at 2000 U/ml CSF-1 for 1day, and then incubated for 48 hours either with additional medium orwith LK, and assayed for TU5 killing as described above.

The results are shown in FIG. 6. While CSF-1 showed marked enhancementof toxicity to TU5, neither CSF-G nor CSF-GM had any effect.

Stimulation of Murine Antiviral Activity

Adherent murine thioglycolate-elicited mcarophages were incubated withCSF-1 for 3 days and infected with VSV overnight. Polymyxin B was addedto test samples to block the LPS induction of interferon. The followingtable shows crystal violet staining of cells remaining adherent.

                  TABLE 3                                                         ______________________________________                                                      Crystal Violet                                                                  -Polymyxin B                                                  Treatment       (mean) (S.D.)                                                                             +Polymyxin B                                      ______________________________________                                        Medium/No VSV    .158 ± .019                                               Medium + VSV    .0583 ± .02                                                                             .049 ± .009                                   CSF-1625 U/ml + VSV                                                                            .139 ± .018                                                                           .177 ± .04                                     1250 + VSV      .167 ± .06                                                                             .205 ± .07                                     2500 + VSV      .160 ± .06                                                                             .219 ± .04                                     5000 + VSV      .150 ± .03                                                                             .202 ± .06                                     ______________________________________                                    

CSF-1 treated cells, therefore, showed protection of the macrophageagainst VSV.

E.7 Formulation of CSF-1

The recombinantly produced human CSF-1 may be formulated foradministration using standard pharmaceutical procedures. OrdinarilyCSF-1 will be prepared in injectable form, and may be used either as thesole active ingredient, or in combination with other proteins or othercompounds having complementary or similar activity. Such other compoundsmay include alternate antitumor agents such as adriamycin, orlymphokines, such as IL-1, -2, and -3, alpha-, beta-, andgamma-interferons and tumor necrosis factor. The effect of the CSF-1active ingredient may be augmented or improved by the presence of suchadditional components. As described above, the CSF-1 may interact inbeneficial ways with appropriate blood cells, and the compositions ofthe invention therefore include incubation mixtures of such cells withCSF-1, optionally in the presence of additional lymphokines. Either thesupernatant fractions of such incubation mixtures, or the entire mixturecontaining the cells as well, may be used.

F. Murine CSF-1

An intronless DNA sequence encoding murine CSF-1 is prepared using amurine fibroblast cell line which produces large amounts of CSF-1. TheL-929 line, obtainable from ATCC, is used as a source for mRNA in orderto produce a cDNA library. Using oligomeric probes constructed on thebasis of the known murine N-terminal and CNBr-cleaved internal peptidesequence, this cDNA library is probed to retrieve the entire codingsequence for the murine form of the protein. Murine CSF-1 is believed tobe approximately 80% homologous to the human material because of thehomology of the N-terminal sequences, the ability of both human andmurine CSF-1 preparations to stimulate macrophage colonies from bonemarrow cells, and limited cross-reactivity with respect to radioreceptorand radioimmunoassays (Das, S. K., et al, Blood (1981) 58: 630).

F.1. Protein Purification

Murine CSF-1 was purified by standard methods similar to those that aredisclosed by Stanley, E. R. et al, J Immunol Meth (1981) 42: 253-284 andby Wang, F. F., et al, J Cell Biochem (1983) 21: 263-275 of SDS gelelectrophoresis as reviewed by Hunkapiller, M. W., et al, Science (1984)226: 304.

Amino acids 1-39 of the murine sequence were obtained, taking advantageof cyanogen bromide cleavage at position 10 to extend the degradationprocedure. An internal cleavage fragment from the mouse protein was alsoobtained and sequenced.

Overall composition data for the mouse protein were also obtained asshown below. These data show correct relative mole % for those aminoacids showing good recoveries; however the numbers are not absolute, ashistidine and cysteine were not recovered in good yield.

    ______________________________________                                        Amino Acid     mole %   residues/125                                          ______________________________________                                        Asp            20.1     25.1                                                  Glu            20.0     25.0                                                  His            --       --                                                    Ser            6.0      7.5                                                   Thr            5.9      7.4                                                   Gly            5.4      6.8                                                   Ala            6.8      8.5                                                   Arg            3.0      3.8                                                   Pro            6.7      8.4                                                   Val            5.3      6.6                                                   Met            1.1      1.4                                                   Ile            3.9      4.9                                                   Leu            8.5      10.6                                                  Phe            6.0      7.5                                                   Lys            3.5      4.4                                                   Tyr            4.1      5.1                                                   ______________________________________                                    

The conversion to residues/125 was based on an approximation of sequencelength from molecular weight.

F.2. Preparation of Murine CSF-1 cDNA

The amino acid sequence 5-13 of the murine CSF-1 and the internalsequence were used as a basis for probe construction.

Three sets of oligomers corresponding to the murine sequence wereprepared. One sequence was prepared to encode "region A"--i.e., aminoacids 9-13; another was prepared to "region B"--i.e., amino acids 5-9,as shown in FIG. 2; a third to encode positions 0-6 of an internalsequence, "region C". Because of codon redundancy, each of these classesof oligomers is highly degenerate.

Thus, 15-mers constructed on the basis of region A number 48; 14-mersconstructed on the basis of region B (deleting the last nucleotide ofthe codon for histidine) also number 48; 20-mers constructed on thebasis of region C number 32. Alternatively stated, a 15-mer constructedso as to encode region A may have a mismatch in four of the fifteenpositions; a particular 14-mer constructed with respect to region B mayhave a mismatch in six positions; a particular 20-mer constructed withrespect to region C may have a mismatch in five positions.

As described below, by suitable protocol design, an enriched messengerRNA fraction may be found for the production of the desired enrichedmurine cDNA library, and the precisely correct oligomers for use asprobes also ascertained.

Totla messenger RNA is extracted and purified from murine L-929 cells.Murine L-929 cells are cultured for 8 days on DME medium and thenharvested by centrifugation. The total cytoplasmic ribonucleic acid(RNA) was isolated from the cells by the same protocol as set forthabove for MIAPaCa mRNA.

The mRNA is fractionated on gels for Northern blot as described inparagraph C.3. The 15-mer sequences corresponding to region A aredivided into four groups of twelve each. Each of these groups was usedto hybridize under low stringency both to control and to murine L-929mRNA slabs and the resulting patterns viewed by radioautography. Underthe low stringency conditions employed, hybridization occurs tofractions not containing the proper sequence, as well as those that do.Also, because the control cell line is different from that of the L-929line in ways other than failure to produce CSF-1, hybridization occursin a number of size locations not related to CSF-1 in the L-929 cellgels which are not present in the controls.

Comparable sets of control and L-929 gels are probed with segregants ofthe 48 14-mers representing region B and segregants of the 32 20-mersrepresenting region C. Only the bands of messenger RNA which hybridizeexclusively in the L-929 slabs for either regions A or B, and C probesare then further considered.

The RNA band which continues to bind to one of the A region 15-mersmixture or one of the region B 14-mers mixture and one of the region C20-mer mixture under conditions of increasingly higher stringency isselected.

When the correct mRNA band is found, each of the groups of region A15-mers is used to probe at various stringency conditions. The groupbinding at highest stringency presumably contains the correct 15-merexactly to complement the mRNA produced. The correct 15-mer isascertained by further splitting the preparation until a single oligomeris found which binds at the highest stringency. A similar approach isused to ascertain the correct 14-mer or 20-mer which binds to region Bor C. These specific oligomers are then available as probes in a murinecDNA library which is prepared from the enriched mRNA fraction.

The mRNA fraction identified as containing the coding sequence for CSF-1is then obtained on a preparative scale. In this preparation, the polyA⁺ mRNA was fractionated on a sucrose gradient in 10 mM Tris-HCl, pH7.4, 1 mM EDTA, and 0.5% SDS. After centrifugation in a Beckman SW40rotor at 30,000 rpm for 17 hr, mRNA fractions are recovered from thegradient by ethanol precipitation. RNA fractions recovered from thegradient were each injected into Xenopus oocytes in a standardtranslation assay and the products assayed for CSF-1 usingradioimmunoassay with antibodies raised against murine CSF-1. Fractionsfor which positive results were obtained were pooled and used toconstruct the cDNA library. These same fractions hybridize to theoligomeric probes.

Other methods of preparing cDNA libraries are, of course, well known inthe art. One, now classical, method uses oligo dT primer, reversetranscriptase, tailing of the double stranded cDNA with poly dG, andannealing into a suitable vector, such as pBR322 or a derivativethereof, which has been cleaved at the desired restriction site andtailed with poly dC. A detailed description of this alternate method isfound, for example, in U.S. Ser. No. 564,224, filed Dec. 20 1983, nowU.S. Pat. No. 4,518,584 and assigned to the same assignee, incorporatedherein by reference.

In the method used here, the enriched mRNA (5 μg) is denatured bytreatment with 10 mM methyl mercury at 22° C. for 5 min and detoxifiedby the addition of 100 mM 2-mercaptoethanol (Payvar, F., et al, J. BiolChem (1979) 254: 7636-7642). Plasmid pcDV1 is cleaved with KpnI, tailedwith dTTP, and annealed to the denatured mRNA. This oligo dT primed mRNAis treated with reverse transcriptase, and the newly synthesized DNAstrand tailed with dCTP. Finally, the unwanted portion of the pcDV1vector is removed by cleavage with HindIII. Separately, pL1 is cleavedwith PstI, tailed with dGTP, cleaved with HindIII, and then mixed withthe poly T tailed mRNA/cDNA complex extended by the pcDV1 vectorfragment, ligated with E. coli ligase and the mixture treated with DNApolymerase I (Klenow) E. coli ligase, and RNase H. The resulting vectorsare transformed into E. coli K12 MM294 to Amp^(R).

The resulting cDNA library is then screened using the oligomer probesidentified as complementary to the mRNA coding sequence as describedabove. Colonies hybridizing to probes from regions A or B and C arepicked and grown; plasmid DNA isolated, and plasmids containing insertsof sufficient size to encode the entire sequence of CSF-1 isolated. Thesequence of the insert of each of these plasmids is determined, and aplasmid preparation containing the entire coding sequence includingregions A and B at the upstream portion is designated pcMCSF.

F.3 Expression of Murine CSF-1 DNA

In a manner similar to that set forth above for the human cDNA, themurine cDNA is tested for transient expression in COS cells, and usedfor expression in stably transformed CV-1. In addition, the appropriateHindIII/ATG encoding sequences are inserted upstream of the matureprotein by mutagenesis and the coding sequences inserted into pPLOP orpTRP3 for procaryotic expression.

G. Host Vectors

pPLOP is a host expression vector having the P_(L) promoter and N generibosome binding site adjacent a HindIII restriction cleavage site, thuspermitting convenient insertion of a coding sequence having an ATG startcodon preceded by a HindIII site. The backbone of this vector is atemperature-sensitive high copy number plasmid derived from pCS3. pPLOPwas deposited at ATCC on Dec. 18 1984, and has accession number 39947.

pTRP3 is a host expression vector containing a trp promoter immediatelyupstream of a HindIII restriction site, thus permitting insertion of acoding sequence in a manner analogous to that above for pPLOP. Thebackbone vector for pTRP3 is pBR322. pTRP3 was deposited with ATCC onDec. 18 1984, and has accession number 39946.

Construction of pPLOP

Origin of Replication

pCS3 provides an origin of replication which confers high copy number ofthe pPLOP host vector at high temperatures. Its construction isdescribed extensively in U.S. Ser. No. 541,948, filed Oct. 14 1983,incorporated herein by reference. pCS3 was deposited June 3 1982 andassigned ATCC number 39142.

pCS3 is derived from pEW27 and pOP9. pEW27 is described by E. M. Wong,Proc Natl Acad Sci (USA) (1982) 79: 3570. It contains mutations near itsorigin of replication which provide for temperature regulation of copynumber. As a result of these mutations replication occurs in high copynumber at high temperatures, but at low copy number at lowertemperatures.

pOP9 is a high copy number plasmid at all temperatures which wasconstructed by inserting into pBR322 the EcoRI/PvuII origin containingfragment from Col El type plasmid pOP6 (Gelfand, D., et al, Proc NatlAcad Sci (USA) (1978) 75: 5869). Before insertion, this fragment wasmodified as follows: 50 μg of pOP6 was digested to completion with 20units each BamHI and SstI. In order to eliminate the SstI 3' protrudingends and "fill in" the BamHI 5' ends, the digested pOP6 DNA was treatedwith E. coli DNA polymerase I (Klenow in a two-stage reaction first at20° C. for elimination of the 3' SstI protruding end and then at 9° C.for repair at the 5' end. The blunt ended fragment was digested and 0.02pmole used to transform competent DG75 (O'Farrell, P., et al, JBacteriology (1978) 134: 645-654). Transformants were selected on Lplates containing 50 μ/ml ampicillin and screened for a 3.3 kb deletion,loss of an SstI site, and presence of a newly formed BamHI site.

One candidate, designated pOP7, was chosen and the BamHI site deleted bydigesting 25 μg of pOP7 with 20 units BamHI, repairing with E. coli DNApolymerase I fragment (Klenow), and religating with T4 DNA ligase.Competent DG75 was treated with 0.1 μg of the DNA and transformantsselected on L plates containing 50 μg/ml ampicillin. Candidates werescreened for the loss of the BamHI restriction site. pOP8 was selected.To obtain pOP9 the AvaI(repaired)/EcoRI Tet^(R) fragment from pBR322 wasprepared and isolated and ligated to the isolated PvuII(partial)/EcoRI3560 bp fragment from pOP8.

Ligation of 1.42 kb EcoRI/AvaI(repair) Tet^(R) (fragment A) and 3.56 kbEcoRI/PvuII Amp^(R) (fragment B) used 0.5 μg of fragment B and 4.5 μg offragment A in a two-stage reaction in order to favor intermolecularligation of the EcoRI ends.

Competent DG75 was transformed with 5 μl of the ligation mixture, andtransformants were selected on ampicillin (50 μg/ml) containing plates.pOP9, isolated from Amp^(R) Tet^(r) transformants, showed high copynumber, colicin resistance, single restriction sites for EcoRI, BamHI,PvuII, HindIII, 2 restriction sites for HincII, and the appropriate sizeand HaeIII digestion pattern.

To obtain pCS3, 50 μg pEW27 DNA was digested to completion with PvuIIand the EcoRI. Similarly, 50 μg of pOP9 was digested to completion withPvuII and EcoRI and the 3.3 kb fragment was isolated.

0.36 μg (0.327 pmoles) pEW27 fragment and 0.35 μg (0.16 pmoles) pOP9fragment were ligated and used to transform E. coli MM294. Amp^(R)Tet^(R) transformants were selected. Successful colonies were initiallyscreened at 30° C. and 41° C. on beta-lactamase assay plate and then forplasmid DNA levels following growth at 30° C. and 41° C. A successfulcandidate, designated pCS3, was confirmed by sequencing.

Preparation of the P_(L) N_(RBS) Insert

The DNA sequence containing P_(L) phage promoter and the ribosomebinding site for the N-gene (N_(RBS)) was obtained from pFC5, andultimately from a derivative of pKC30 described by Shimatake andRosenberg, Nature (1981) 292: 128. pKC30 contains a 2.34 kb fragmentfrom lambda phage cloned into the HindIII/BamHI vector fragment frompBR322. The P_(L) promoter and N_(RBS) occupy a segment in pKC30 betweena BglII and HpaI site. The derivative of pKC30 has the BglII siteconverted to an EcoRI site.

The BglII site immediately preceding the P_(L) promoter was convertedinto an EcoRI site as follows: pKC30 was digested with BglII, repairedwith Klenow and dNTPs and ligated with T4 ligase to an EcoRI linker(available from New England Biolabs) and transformed into E. coli K12strain MM294 lambda⁺. Plasmids were isolated from Amp^(R) Tet^(R)transformants and the desired sequence confirmed by restriction analysisand sequencing. The resulting plasmid, pFC3, was double-digested withPvuI and HpaI to obtain an approximately 540 bp fragment isolated andtreated with Klenow and dATP, followed by Sl nuclease, to generate ablunt ended fragment with the 3' terminal sequence -AGGAGAA where the-AGGAGA portion is the N_(RBS). This fragment was restricted with EcoRIto give a 347 base pair DNA fragment with 5'-EcoRI (sticky) and HinfI(partial repair, Sl blunt)-3' termini.

To complete pFC5, pβI-Z15 was used to create a HindIII site 3' of theN_(RBS). pβI-Z15 was deposited Jan. 13 1984, ATCC No. 39578, and wasprepared by fusing a sequence containing ATG plus 140 bp of β-IFN fusedto lac Z into pBR322. In pβI-Z15, the EcoRI site of pBR322 is retained,and the insert contains a HindIII site immediately preceding the ATGstart codon of β-IFN. pβI-Z15 was restricted with HindIII, repaired withKlenow and dNTPs, and then digested with EcoRI. The resultingEcoRI/HindIII (repaired) vector fragment was ligated with theEcoRI/HinfI (repaired) fragment above, and the ligation mixture used totransform MC1000-39531. Transformants containing the successfulconstruction were identified by ability to grow on lactose minimalplates at 34° but not at 30°. (Transformations were plated on X-gal-Ampplates at 30° and 34° and minimal-lactose plates at 30° and 34°.Transformants with the proper construction are blue on X-gal-Amp platesat both temperatures, but on minimal lactose plates, grow only at 34°.)The successful construct was designated pFC5.

Completion of pPLOP

pCS3 was then modified to provide the P_(L) and N_(RBS) controlsequences. pCS3 was digested with HindIII, and then digested with EcoRI.The vector fragment was ligated with an isolated EcoRI/HindIII from pFC5containing the P_(L) N_(RBS) and transformed into E. coli MM294. Thecorrect construction of isolated plasmid DNA was confirmed byrestriction analysis and sequencing and the plasmid designated pPLOP.

Preparation of pTRP3

To construct the host vector containing the trp control sequences behinda HindIII site, the trp promoter/operator/ribosome binding sitesequence, lacking the attenuator region, was obtained from pVH153,obtained from C. Yanofsky, Stanford University. Trp sequences areavailable in a variety of such plasmids known in the art. pVH153 wastreated with HhaI (which cuts leaving an exposed 3' sticky end just 5'of the trp promoter) blunt ended with Klenow, and partially digestedwith TaqI. The 99 bp fragment corresponding to restriction at the TaqIsite, 6 nucleotides preceding the ATG start codon of trp leader, wereisolated, and then ligated to EcoRI (repair)/ClaI digested, pBR322 toprovide pTRP3.

On Apr. 2 1985, Applicants have deposited with the American Type CultureCollection, Rockville, MD, USA (ATCC) the phage pHCSF-1 in E. coli DG98,accession no. 40177. On May 21 1985, pHCSF-1a, designated CMCC 2312 inthe Cetus collection and pHCSF-1 λ Charon 4A for deposit, was depositedwith ATCC and has accession no. 40185. On June 14 1985, CSF-17 in E.coli MM294, designated CMCC 2347, was deposited with ATCC and hasaccession no. 53149. In addition, the folllowing deposits were made withATCC on the date of June 19 1986:

    ______________________________________                                        Plasmid        CMCC No.  ATCC No.                                             ______________________________________                                        pCSF-asp59     2705      67139                                                pCSF-gln52     2708      67140                                                pCSF-pro52     2709      67141                                                pCSF-Bam       2710      67142                                                pCSF-BamBcl    2712      67144                                                pCSF-Gly150    2762      67145                                                ______________________________________                                    

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture for 30 years fromdate of deposit. The deposits will be made available by ATCC under theterms of the Budapest Treaty, and subject to an agreement betweenApplicants and ATCC which assures permanent and unrestrictedavailability upon issuance of the pertinent U.S. patent. The Assigneeherein agrees that if the culture on deposit should die or be lost ordestroyed when cultivated under suitable conditions, it will be promptlyreplaced upon notification with a viable specimen of the same culture.Availability of the deposits is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

These deposits were made for the convenience of the relevant public anddo not constitute an admission that a written description would not besufficient to permit practice of the invention or an intention to limitthe invention to these specific constructs. Set forth hereinabove is acomplete written description enabling a practitioner of ordinary skillto duplicate the constructs deposited and to construct alternative formsof DNA, or organisms containing it, which permit practice of theinvention as claimed.

The scope of the invention is not to be construed as limited by theillustrative embodiments set forth herein, but is to be determined inaccordance with the appended claims.

We claim:
 1. An isolated DNA sequence which encodes for human CSF-1polypeptide or a murine CSF-1 polypeptide, wherein said polypeptidestimulates the formation of primarily macrophage colonies in the invitro CSF-1 assay.
 2. The DNA sequence of claim 1 which encodes humanCSF-1.
 3. The DNA sequence of claim 2 wherein the human CSF-1 has anamino acid sequence substantially equivalent to that of native humanmCSF-1.
 4. The recombinant DNA sequence of claim 2 wherein the aminoacid sequence of the human CSF-1 is related to that of mCSF-1 by thedeletion or conservative substitution of one or more amino acidsresiding between positions 150 and 224 inclusive of mCSF-1 as shown inFIG.
 5. 5. The DNA sequence of claim 2 wherein the human CSF-1 has anamino acid sequence which is related to that of mCSF-1 by the deletionor conservative substitution of one or more of the amino acids atpositions 51 and 52 and/or positions 191, 192 and 193 of mCSF-1 as shownin FIG.
 5. 6. The DNA sequence of claim 2 wherein the human CSF-1 has anamino acid sequence which is related to that of mCSF-1 by the deletionor conservative substitution of one or more of the amino acids atpositions 15-20 and/or positions 75-84 of mCSF-1 as shown in FIG.
 5. 7.The DNA sequence of claim 2 wherein the human CSF-1 has an amino acidsequence which is related to that of mCSF-1 by the deletion orsubstitution of the tyrosine residue at position 59 of mCSF-1.
 8. TheDNA sequence of claim 2 wherein the human CSF-1 has an amino acidsequence which is selected from the group consisting of mCSF-1, ∇₁₅₈₋CSF-1, ∇₁₅₀₋ CSF-1, gln₅₂ CSF-1, and asp₅₉ CSF-1.
 9. The DNA sequence ofclaim 1 wherein the CSF-1 has the amino acid sequence of native murineCSF-1.
 10. The DNA sequence of claim 2 which encodes a protein of theamino acid sequence encoded in pHCSF-1a or in CSF-17.
 11. A replicativecloning vector which comprises the DNA sequence encoding for a humanCSF-1 polypeptide or a murine CSF-1 polypeptide, wherein saidpolypeptide stimulates the formation of primarily macrophage colonies inthe in vitro CSF-1 assay, and a replicon operative in a unicellularorganism.
 12. The vector of claim 11 wherein the sequence encoding CSF-1encodes a protein which has an amino acid sequence substantiallyequivalent to that in human mCSF-1.
 13. The vector of claim 12 whereinthe sequence encoding CSF-1 encodes a protein which has an amino acidsequence selected from the group consisting of mCSF-1, ∇₁₅₈₋ CSF-1,∇₁₅₀₋ CSF-1, gln₅₂ CSF-1, and asp₅₉ CSF-1.
 14. The vector of claim 11wherein the sequence encoding CSF-1 encodes a protein of the amino acidsequence encoded in pHCSF-1a or pcCSF-17.
 15. An expression system whichcomprises the DNA of claim 1 operably linked to suitable controlsequences.
 16. The expression system of claim 15 wherein the recombinantDNA sequence encodes human CSF-1 which has an amino acid sequencesubstantially equivalent to that of human mCSF-1.
 17. The expressionsystem of claim 15 wherein the recombinant DNA sequence encodes humanCSF-1 which has an amino acid sequence selected from the groupconsisting of mCSF-1, ∇₁₅₈₋ CSF-1, ∇₁₅₀₋ CSF-1, gln₅₂ CSF-1, and asp₅₉CSF-1.
 18. The expression system of claim 15 wherein the recombinant DNAsequence encoding CSF-1 comprises pHCSF-1a or pcCSF-17.
 19. Theexpression system of claim 15 disposed in a vector capable ofreplication in suitable host cells.
 20. The expression system of claim16 disposed in a vector capable of replication in suitable host cells.21. Recombinant host cells transformed with the expression system ofclaim
 15. 22. Recombinant host cells transformed with the expressionsystem of claim
 16. 23. A method of produciang recombinant CSF-1 whichcomprises culturing the cells of claim 21 under conditions effective forthe production of said CSF-1.
 24. A method of producing recombinantCSF-1 which comprises culturing the cells of claim 22 under conditionseffective for the production of said CSF-1.
 25. The DNA of claim 2 whichis disposed in clone pHCSF-1a.
 26. The DNA of claim 2 which is disposedin clone pcCSF-17.
 27. An isolated DNA sequence which encodes for ahuman CSF-1 polypeptide wherein the CSF-1 has an N-terminal amino acidsequence comprising:Glu-Glu-Val-Ser-Glu-Tyr-Cys-Ser-His-Met-lle-Gly-Ser-Gly-His-Leu-Gln-Ser-Leu-Gln-Arg-Leu-lle-Asp-Ser-Gln-Met-Glu-Thr-Ser-Cys-Gln-lle-Thr-Phe-Glu-Phe-Val-Asp-Gln-Glu-Gln-Leuand wherein said polypeptide stimulates the formation of primarilymacrophage colonies in the in vitro CSF-1 assay.
 28. An isolated DNAsequene which encodes for a murine CSF-1 polypeptide wherein the CSF-1has an N-terminal amino acid sequence comprisng:Lys-Glu-Val-Ser-Glu-His-Cys-Ser-His-Met-Ile-Gly-Asn-Gly-His-Leu-Lys-Val-Leu-Gln-Gln-Leu-Ile-Asp-Ser-Gln-Met-Glu-Thr-Ser-and wherein said polypeptide stimulates the formation of primarilymacrophage colonies in the in vitro CSF-1 assay.
 29. A replicativecloning vector which comprises the DNA sequence of claim 27 or 28 and areplicon operative in a unicellular organism.
 30. An expression systemwhich comprises the DNA sequence of claim 27 or 28 operably linked tosuitable control sequences.
 31. Recombinant host cells transformed withan expression system which comprises the DNA sequence of claim 27 or 28operably linked to suitable control sequences.
 32. A method of producingrecombinant CSF-1 which comprises culturing recombinant host cellstransformed with an expression system which comprises the DNA sequenceof claim 23 operably linked to suitable control sequences underconditions effective for the production of said CSF-1.
 33. A method ofproducing recombinant CSF-1 which comprises culturing recombinant hostcells transformed with an expression system which comprises the DNAsequence of claim 28 operably linked to suitable control sequences underconditions effective for the production of said CSF-1.